Highly water-soluble metalloproteinase inhibitors

ABSTRACT

New compounds of general formulae (III), (IV) and (V).

This application is a divisional of application Ser. No. 08/945,356,filed on Oct. 24, 1997 now U.S. Pat. No. 6,147,114. Application Ser. No.08/945,356 is the national phase of PCT International Application No.PCT/JP96/01135 filed on Apr. 25, 1996 under 35 U.S.C. § 371. The entirecontents of each of the above-identified applications are herebyincorporated by reference.

SPECIFICATION

1. Field of Industrial Application

The invention relates to new highly water-soluble compounds, whichinhibit matrix metalloproteinases (MMPs) derived from vertebrates and/ortumor necrosis factor-α (TNF-α) converting enzyme, preparationintermediates thereof and a process for the preparation thereof.

2. Background Art

MMPs which belong to endo proteinases containing zink are involved inthe decomposition of an extra-cellular matrix in a connective tissue. Upto now, it is known that there exist ten-odd MMPs, and the expression ofthese enzymes are strictly controlled in healthy volunteers. However, anabnormal aggravation of MMPs is observed in cases such as chronicrheumatoid arthritis, osteoarthritis, periodental disease, cornealulcer, various kinds of bullosis (epidermolisis bullosa hereditaria,epidermolisis bullosa acquisita, porphylia cutanea tarda, bullospenphigoid, pemphigus vulgaris), intractable skin ulcer (bedsore, skinulcer in radiotherapy, skin ulcer in diabetes mellitus, skin ulcer inarteriosclerotic obliteration), wound (external injury or burn),osteoporosis, cancer metasis and the like, and these are considered toparticipate in the destruction of the extra-cellular matrix. [D. Brownet al., Current Eye Research, 12, 571(1993)/Y. Okada et al., VirchowsArchiv. B, Cell Pathol., 59, 305(1990)/W. G. Stetler-Stevenson, CancerMetastasis Reviews, 9,289(1990)/H. Birkedal-Hansen et al., CriticalReviews in Oral Biology and Medicine, 4(2),197(1993)] On the contrary,TNF-α is produced as a membrane-bound type precursor of molecular weight26K, and in case of the excess of an extra-cellular release isconsidered the occurrence of diseases such as sepsis, chronic rheumatoidarthritis or the like. Recently, it was reported that the enzyme (TNF-αconverting enzyme) inducing the release of TNFα was a metalloproteinasewhose activity was controlled by a MMPs inhibitor. [A. J. H. Gearing etal., Journal of Leukocyte Biology, 57, 774(1995), K. M. Mohler et al.,Nature, 370, 218(1994), G. M. NcGeehan et al., Nature, 370, 558(1994)].

Accordingly, in the above diseases, inhibiting the action of theseenzymes becomes an effective method of therapy. However, as thecompounds having MMPs inhibitory activities are known the compoundsdivided into four families which are phosphonic acid derivatives,hydroxamic acid derivatives, derivatives having mercapto group andderivatives having carboxyl group. Especially, on the hydroxamic acidderivatives are proposed compounds having various skeletons (see U.S.Pat. No. 4,599,361, EP, 575844 A2, U.S. Pat. No. 5,412,145, WO,92/13831, U.S. Pat. No. 5,183,900, WO, 94/02447, EP, 606046 A1 and GB2268933 A), and many of these compounds have highly inhibitoryactivities for various kinds of MMPs. However, each of these compoundsis poor in its water-solubility and their administration methods arelimited. For example, in case of applying these compounds as injections(aqueous solution), medicaments of high concentration cannot beprepared. Furthermore, in case of administering injections into joint,if there is a particle not less than 50 μm, the occurrence of a synovialinflammation is known, therefore, it becomes a necessary condition for acompound to be completely dissolved state. In the known compounds, suchan administration is impossible, and it is a present situation that theyare not effectively utilized as therapeutic agents.

DISCLOSURE OF THE INVENTION

The inventors gave attention to the hydroxamic acid derivatives havinghighly inhibitory activities, and as the result of making an extensivestudies to enhance the availability, we accomplished the invention byfinding out new hydroxamic acid derivatives whose water-solubility wasdramatically increased compared with that of the above compounds' group.

Accordingly, the invention provides compounds of general formula (I)

(wherein R¹ is a hydrogen atom, or a hydroxyl, aryl(C₁-C₆)alkylene or—A—SOn—B group (A is a (C₁-C₆) alkylene group; B is a (C₁-C₆) alkyl,(C₁-C₆) acyl, aryl or heterocyclyl groups; n is 0, 1 or 2), R² is ahydrogen atom, or a (C₁-C₆) alkyl, (C₁-C₆) alkyloxy or (C₁-C₆) alkylthiogroups, R³ and R⁴ are identical or different, representing a hydrogenatom, or a (C₁-C₆) alkyl, aryl or aryl(C₁-C₆)alkylene groups, R⁵ is aY—C″ or C″ group (Y is a (C₁-C₆) alkylene group, an oxygen atom, animino or (C₁-C₆) alkyleneimino groups, C″ is a sulfonic acid, phosphonicacid, amidino, (C₁-C₆) acyl, acylimidoyl, diphosphonomethine ordicarboxymethine groups), and R⁶ is a hydrogen atom, or a nonsubstitutedor substituted benzyl, trialkylsilyl, tert-butyldiphenylsilyl,tetrahydropyranyl or tert-butyl groups) or stereoisomers thereof, andpharmaceutically acceptable salts thereof and solvates thereof, andmetalloproteinase inhibitors which comprise one or more compoundsselected from those compounds as effective ingredients and inhibitmatrix metalloproteinases (MMPs) and/or TNF-α converting enzyme.Further, the invention provides preparation intermediates to obtain thecompounds of the above formula (I) and process for the preparation.

In the following, the invention will be explained in detail.

In the compounds represented by the general formula (I) according to theinvention are included compounds described below.

The sulfonic acid group indicates —SO₃H, and the sulfuric acid group is—OSO₃H. The phosphonic acid group indicates —PO₃H₂, and phosphate is—OPO₃H₂. The amidino group indicates —C(═NH)NH₂, and the guanido groupdoes —NH—C(═NH)NH₂. The aminomethylene group indicates —CH₂NH₂ and theguanidomethylene group —CH₂—NH—C(═NH)NH₂. The acetamidomethylene groupindicates —CH₂NH—COCH₃, the acetimidoyliminomethylene group—CH₂—NH—C(═NH)CH₃, the propionimidoyliminomethylene group—CH₂—NH—C(═NH)CH₂CH₃, the benzimidoyliminomethylene group—CH₂—NH—C(═NH)C₆H₅, the diphosphonomethine group —CH[PO(OH)₂]₂ and thedicarboxymethine group —CH(CO₂H)₂, respectively.

The (C₁-C₆) alkyl group indicates an alkyl group of straight chain orbranched chain containing 1-6 carbon atoms, including methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl and hexylgroups, etc. The (C₁-C₆) alkylene group indicates —(CH₂)_(n)—(n=1-6).The imino group indicates —NH—.

The (C₁-C₆) alkyleneimino group indicates —(CH₂)_(n)—NH—(n=1-6), whichis preferably (C₁-C₃) alkyleneimino group and more preferably —CH₂—NH—.

The (C₁-C₆) acyl group indicates a alkylacyl group of straight chain orbranched chain containing 1-6 carbon atoms, including formyl, acetyl,n-propanoyl and n-butanoyl groups, etc.

The acylimidoyl group indicates —C(═NH)—[(C₁-C₆)alkyl] or—C(═NH)-(aryl).

The heterocyclyl group indicates a cyclic skeleton saturated orunsaturated, having at least one of hetero atoms such as sulfur, oxygenor nitrogen atoms, etc. Illustrative of the preferable heterocyclylgroup are, for example, thienyl, thiazolyl, imidazolyl or pyridylgroups, etc.

The aryl group indicates aromatic rings such as phenyl, naphthyl oranthracenyl groups, etc., which can be substitued.

The substituents on the aryl group include (C₁-C₆) alkyl, (C₁-C₆) acyl,hydroxyl, amino and carboxyl groups, a halogen atom, etc., and in casehaving two or more substituents, can be a combination of these.

The halogen atom indicates fluorine, chlorine, bromine or iodine atoms.

Additionally, as the salts of compounds of the general formula (I) areincluded inorganic salts consisting of alkaline metals such as sodiumand potassium, etc., alkaline earth metals such as magnesium andcalcium, etc., or mineral acids such as hydrochloric acid andhydrobromic acid, etc., and ammonia consisting of organic salts,morpholine, piperidine, dimethylamine, diethylamine, acetic acid, citricacid, oxalic acid, etc. Further, the solvate is, for example, a hydrate.

Since there exist three asymmetric carbon atoms in the compoundsrepresented by the above general formula (I), all the diasteromers ofthe general formula (I) and the racemates which are a mixture thereofare included.

In the following are explained in detail showing the synthesis-routescheme of compounds of the above general formula (I) according to theinvention and of preparation intermediates thereof. In the followingdescription, the compounds are added with number one by one according tothe method used conventionally in chemical literature, and the compoundsare represented by their numbers. First, the process of preparing theintermediate (II) is explained.

Synthetic route of intermediate (II)

(In the above synthetic route, R¹ and R² have the same meaning describedabove, and R⁷, R⁸, R⁹ and R¹⁰ are identical or different, representing(C₁-C₆) alkyl, benzyl, substituted benzyl, phenacyl or2,2,2-trichloroethyl groups, and X represents halogen atom or oxogroup.)

The route, in which the preparation intermediate (II) is synthesizedfrom the general formula (VIII) as a starting material, is preferable inthe case that R¹ is hydrogen atom, —A—SO_(n)—B— (A is the (C₁-C₆)alkylene group; B is (C₁-C₆) alkyl, (C₁-C₆) acyl, aryl or heterocyclylgroups; n is 0, 1 or 2.) or aryl (C₁-C₆)alkylene group.

The general formula (XI) can be obtained by reactingα-halogenocarboxylate (VIII), in which X is a halogen atom, and theformula (IX), being the anionized malonic acid diester (R² has the samemeaning described above, and is preferably the (C₁-C₅) alkyl group, morepreferably iso-propyl group. The halogen atom is fluorine, chlorine,bromine or iodine atoms, though, preferably a bromine atom.

As a base used in the anionization of malonic acid diester can be usedpotassium tert-butoxide, sodium hydride, lithium diisopropylamide (LDA),lithium bis(trimethylsilyl)amide, alcholate or the like, though,preferably sodium hydride or potassium tert-butoxide.

As an inactive organic-solvent for the reaction solvent can be usedn-hexane, tetrahydrofuran (THF), benzene, toluene, dimethylformamide(DMF), dimethylacetamide (DMAc), dichloromethane, chloroform, or thelike, though, preferably DMF.

The reaction temperature is usually from −78 to 50° C., preferably from0 to 20° C. The reaction time is different depending on a startingmaterial, solvent, reaction temperature, etc., though, from 30 min. to 4hours, usually from 1 to 2 hours.

Subsequently, for the reaction with α-halogenocarboxylate (VIII), as thereaction solvent is used an inactive organic-solvent such as n-hexane,THF, benzene, toluene, DMF, DMAc, dichloromethane, chloroform, or thelike, though, preferably DMF. The reaction temperature is usually from−10 to 50° C., preferably from −5 to 0° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 2 to 24 hours,preferably from 10 to 20 hours.

The compound of the general formula (XIII) can be prepared by lettingthe general formula (XI) to the anionized substance (XII) and reactingit with halogenated aryl-(C₁-C₆) alkylene or halogenated alkenyl, ifdesired, followed by hydrogenation (as aryl-(C₁-C₆) is alkylenepreferably phenyl(C₁-C₅)alkylene group, more preferablyphenyltrimethylene group. As alkenyl group preferable cinnamyl group(C₆H₅—CH═CH—CH₂—). As the halogen atom can be cited fluorine, chlorine,bromine or iodine atoms, preferably bromine atom.

As the base used in the synthesis of the formula (XII) can beusedpotassium tert-butoxide, LDA, lithiumbis(trimethylsilyl)amide,sodium hydride, alcholate or the like, which are conventionally used inthis kind of reaction, though, preferably sodium hydride.

As the reaction solvent are used an inactive organic-solvent such asn-hexane, THF, benzene, toluene, DMF, DMAc, dichloromethane, chloroform,or the like, though, preferably DMF. The reaction temperature is usuallyfrom −10 to 50° C., preferably from 10 to 30° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 30 min. to 4hours, preferably from 1 to 2 hours.

Following to this, in order to let it react with halogenatedaryl-(C₁-C₆)alkylene or halogenated alkenyl, as the reaction solvent isused an inactive organic-solvent such as n-hexane, THF, benzene,toluene, DMF, DMAc, dichloromethane, chloroform, or the like, though,preferably DMF. The reaction temperature is usually from −10 to 50° C.,preferably from −5 to 10° C. The reaction time is different depending ona starting material, solvent, reaction temperature, etc., though,usually from 2 to 48 hours, preferably from 10 to 20 hours. Further, ifdesired, to get the general formula (XIII) by hydrogenation can be usedcatalysts such as palladium-carbon, platinum or the like as ahydrogenation catalyst, though, preferably palladium-carbon. As thereaction solvent can be used an inactive organic-solvent such asalcohols (methanol, ethanol, etc.), DMF, DMAc or acetic acid, which arenot catalytic poison, or water, preferably methanol or ethanol. Thereaction temperature is usually from 0 to 50° C., preferably from 10 to30° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 1 to 24 hours,preferably from 1 to 6 hours.

The compound of the general formula (XIV) can be prepared by thedeestrification of the general formula (XIII). R⁷, R⁸ and R⁹ have thesame meaning described above, though, in order to get the generalformula (XIV), preferably, R⁷ is tert-butyl group and R⁸ and R⁹ arebenzyl group. For example, in the case that R⁸ and R⁹ are benzyl groupcan be deesterfied by hydrogenation.

As the hydrogenation catalyst can be used catalysts such aspalladium-carbon, platinum, or the like, though, preferablypalladium-carbon. As the reaction solvent can be used an inactiveorganic-solvent such as alcohol (methanol, ethanol, etc.), DMF, DMAc oracetic acid, which are not catalytic poison, or water, preferablymethanol or ethanol. The reaction temperature is usually from 0 to 50°C., preferably from 10 to 30° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 1 to 24 hours,preferably from 1 to 7 hours.

The general formula (XV) can be prepared by deesterifying the generalformula (XI). R⁷, R⁸ and R⁹ have the same meaning described above,though, in order to get the general formula (XV), preferably, R⁷ istert-butyl group and R⁸ and R⁹ are benzyl group. For example, in thecase that R⁸ and R⁹ are benzyl group can be deesterfied byhydrogenation. As the hydrogenation catalyst can be used catalysts suchas palladium-carbon, platinum, or the like, though, preferablypalladium-carbon. As the reaction solvent can be used an inactiveorganic-solvent such as alcohols (methanol, ethanol, etc.), DMF, DMAc oracetic acid, which are not catalytic poison, or water, preferablymethanol. The reaction temperature is usually from 0 to 50° C.,preferably from 10 to 30° C. The reaction time is different depending ona starting material, solvent, reaction temperature, etc., though,usually from 1 to 24 hours, preferably from 1 to 6 hours.

The compound of the general formula (XVI) can be prepared by reactingthe Schiff base, which results from formaline and a secondary amine suchas piperidine, diethylamine, morpholine or the like, with the generalformula (XV), followed by decarboxylation and a simultaneous double bondformation. As the reaction solvent, there is no special restriction ifit does not hinder the proceeding of the reaction and dissolve thestarting material, though, alcohols, DMF, DMAc or the like can be cited,preferably methanol.

The reaction temperature is usually from 0 to 150° C., preferably from10 to 100° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 5 to 24 hours,preferably from 10 to 15 hours.

The general formula (X) can be prepared by reacting the Wittig reactionof α-oxocarboxylate (VIII), in which X is a halogen atom, with aphosphorane derivative (R⁷O₂CCH═PE₃; R⁷ has the same meaning describedabove, E is phenyl or alkyl groups.).

As the reaction solvent, there is no special restriction if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, it is diethyl ether, dioxane, THF, toluene, benzene,dichloromethane, chloroform, DMF or the like, preferably THF,dichloromethane or benzene.

The reaction temperature is usually from −70 to 120° C., preferably from−20 to 30° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 30 min. to 24hours, preferably from 1 to 15 hours.

The synthetic route of the preparation intermediate (II) using thegeneral formula (XVII) as a starting material is preferable in the casethat R¹ is a hydrogen atom or a hydroxyl group.

The general formula (XVIII) can be prepared by reacting the succinicacid derivative with (a) alcohols in the presence of a condensationreagent such as N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCl)or the like, and of N,N-dimethylaminopyridine, or (b) halogenated alkylor halogenated benzyl after converting the formula (XVIII) into salts ofsodium, potassium, cesium or the like, or (c) a complex of thionylchloride and alcohol.

For example, in case of reacting the formula (XVII) with the thionylchloride-alcohol complex (c), alcohols used have no special restriction,which are methanol, ethanol, n-propyl alcohol, isopropyl alcohol,tert-butyl alcohol, phenol, benzyl alcohol, phenacyl alcohol,2,2,2-trichloroethanol or the like (preferably, methanol, ethanol,iso-propyl alcohol, tert-butyl alcohol, and more preferably, iso-propylalcohol), and are used accompanying the reaction solvent.

The reaction temperature is usually from −30 to 10° C., preferably from−10 to 0° C. The reaction time is different depending on a startingmaterial, solvent, reaction temperature, etc., though, usually from 5 to24 hours, preferably from 10 to 15 hours.

The compound of the general formula (XIX) can be prepared by anionizingthe general formula (XVIII) and reacting with halogenated (C₁-C₆)alkylor halogenated (C₁-C₆)alkenyl, if desired, followed by hydrogenation.(As (C₁-C₆) alkyl group preferable is (C₁-C₅) alkyl group, especiallypreferably iso-butyl group. As (C₁-C₆) alkenyl group preferable is(C₁-C₅) alkenyl group, especially preferably 2-methylpropene group(—CH₂—C(═CH₂)CH₃). As the halogen atom can be cited fluorine, chlorine,bromine or iodine atoms, preferably bromine atom.)

As the base used in the anionization of succinic acid diester (XVIII)can be used potassium tert-butoxide, sodium hydride, LDA, lithiumbis(trimethylsilyl)amide, alcholate or the like, which are used in aconventional reaction, though, preferably LDA.

As the reaction solvent are used an inactive organic-solvent such asn-hexane, THF, benzene, toluene, DMF, DMAc, dichloromethane, chloroform,or the like, though, preferably THF. The reaction temperature is usuallyfrom −78 to 0° C., preferably from −70 to −10° C.

The reaction time is different depending on a startingmaterial, solvent,reaction temperature, etc., though, from 30 min. to 24 hours, usuallyfrom 4 to 12 hours.

Following to this reaction, in order to let it react with halogenated(C₁-C₆)alkyl or halogenated (C₁-C₆)alkenyl, as the reaction solvent isused an inactive organic-solvent such as n-hexane, THF, benzene,toluene, DMF, DMAc, dichloromethane, chloroform, or the like, though,preferably THF.

The reaction temperature is usually from −78 to 0° C., preferably from−70 to −10° C. The reaction time is different depending on a startingmaterial, solvent, reaction temperature, etc., though, usually from 2 to48 hours, preferably from 10 to 20 hours. Further, if desired, to getthe general formula (XIX) by hydrogenation can be used catalysts such aspalladium-carbon, platinum or the like as a hydrogenation catalyst,though, preferably palladium-carbon. As the reaction solvent can be usedan inactive organic-solvent such as alcohol (methanol, ethanol, etc.),DMF, DMAc or acetic acid, which are not catalytic poison, or water,preferably methanol. The reaction temperature is usually from 0 to 50°C., preferably from 10 to 30° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 1 to 24 hours,preferably from 1 to 6 hours.

The compound of the general formula (XX) can be prepared by thedeestrification of the general formula (XIX). For example, in the casethat R¹⁰ is iso-propyl group, the deesterification can be done by analkaline hydrolysis.

As the base used in the hydrolysis, there is no special restriction ifit is used as a base in a usual reaction, though, examples are sodiumcarbonate, potassium carbonate, sodium hydroxide, potassium hydroxide,sodium bicarbonate, lithium hydride, sodium hydride or the like,preferably sodium hydroxide or potassium hydroxide.

As the reaction solvent, there is no special restriction if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, it is DMF, DMAc, alcohols, diethyl ether,tetrahydrofuran, dioxane, water, acetone or the like, preferablyalcohols, water, dioxane, acetone or a mixed solvent thereof. Thereaction temperature is usually from −20 to 150° C., preferably from −10to 100° C. The reaction time is usually from 5 min. to 36 hours,preferably from 10 min. to 24 hours.

The synthetic intermediate (II) can be prepared by (a) decarboxylatingthe general formula (XIV), (b) adding the thiol derivative (HS-B, B hasthe same meaning as described above to the butenoic acid derivative ofthe general formula (XVI)), (c) hydrolyzing the diester of the generalformula (XIV) partially or (d) reacting the acid anhydride (XXI) fromthe dicarboxylic acid (XX) with alcohols or the directhalf-esterification. Additionally, it can be prepared by (e)decarboxylating the formula (XV) in the case that R¹ is a hydrogen atomor (f) hydrogenating the formula (X).

(a), (e) In order to decarboxylate the general formula (XIV) or (XV),the reaction is carried out in the presence of a tertiary amine such astriethylamin, N-methylmorpholine, N-ethylmorpholine or the like. As thereaction solvent can be cited an inactive organic-solvent such asn-hexane, toluene, benzene or the like, preferably toluene.

The reaction temperature is usually from 70 to 150° C., preferably from100 to 120° C. The reaction time is different depending on a startingmaterial, solvent, reaction temperature, etc., though, usually from 1 to5 hours, preferably from 2 to 3 hours.

(b) In adding the thiol derivative to the general formula (XVI), thereis no special restriction for the reaction solvent if it does not hinderthe proceeding of the reaction and dissolves a starting material,though, examples cited are alcohols, DMF, DMAc, dichloromethane,chloroform, or the like, preferably methanol. The reaction can becarried out without solvent.

The reaction temperature is usually from 0 to 100° C., preferably from 0to 60° C. The reaction time is different depending on a startingmaterial, solvent, reaction temperature, etc., though, usually from 10hours to 1 month, preferably from 10 hours to 2 weeks.

(c) The partial hydrolysis of the diester (XIX) is carried out using abase in an equivalent mole. The base used has no special restriction ifit is used in a usual reaction, though, it is an inorganic base such assodium hydroxide, potassium hydroxide, sodium carbonate or the like. Thereaction solvent has no special restriction if it does not hinder theproceeding of the reaction and dissolves a starting material, though,examples cited are alcohols, DMF, DMAc, dichloromethane, chloroform, orthe like, preferably methanol. The reaction temperature is usually from−20 to 50° C., preferably from −5 to 5° C. The reaction time isdifferent depending on a starting material, solvent, reactiontemperature, etc., though, usually from 10 min. to 24 hours, preferablyfrom 30 min. to 5 hours.

(d) The reaction for the preparation of the preparation intermediate(II), for example, via the acid anhydride (XXI) from the dicarboxylicacid (XX) can be carried out [J. Org. Chem., 47, 4928 (1982)]. Alcoholsreacting with the acid anhydride (XXI) has no special restriction,though, they are methanol, ethanol, n-propyl alcohol, iso-propylalcohol, tert-butyl alcohol, phenol, benzyl alcohol, phenacyl alcohol,2,2,2-trichloroethanol or the like, preferably, methanol, ethanol,benzyl alcohol or 2,2,2-trichloroethanol.

(f) For preparing the preparation intermediate (II) by hydrogenating thepropene acid derivative (X) preferable is the compound (X) in which R⁷is tert-butyl group and R⁹ is benzyl group.

As the hydrogenation catalyst can be used catalysts such aspalladium-carbon, platinum, or the like, though, preferablypalladium-carbon. As the reaction solvent can be used an inactiveorganic-solvent such as alcohols (methanol, ethanol, etc.), DMF, DMAc oracetic acid, which are not catalytic poison, or water, preferablymethanol or acetic acid. The reaction temperature is usually from 0 to50° C., preferably from 10 to 30° C. The reaction time is differentdepending on a starting material, solvent, reaction temperature, etc.,though, usually from 1 to 24 hours, preferably from 1 to 6 hours.

In the following, the preparation process of the preparationintermediate (III) is explained.

Synthetic route of intermediate (III)

(In the above synthetic route, R³ and R⁴ have the same meaning describedabove, R¹¹ is an aminoprotecting group such as tert-butyloxycarbonyl(Boc), benzyloxycarbonyl (Z) or substituted Z groups, etc., and D hasthe same meaning as that of R⁵ or is a hydrogen atom, or hydroxyl,protected phosphoric acid, protected diphosphonomethine, protecteddicarboxymethine, nitro, amino, protected guanido, protectedguanidomethylene, cyano, aminomethylene or protected amidino groups.)

In the above each intermediate, the timing of converting the compoundswhose D is a hydrogen atom, or hydroxyl, nitro or cyano groups into thecompound having a desirable functional group such as sulfuric acid,sulfonic acid, phosphoric acid, phosphonic acid, protected ornon-protected guanido, amino, aminomethylene, acylimidoylaminomethylene,(C₁-C₆) acylamidoaminomethylene or protected or non-protected amidinogroups, etc., is different depending on the characteristics of eachfunctional group. For example, (a) in case of sulfonic acid group, itcan be prepared by the sulfonation of 4′ position of phenylalanine [seeHelv. Chim. Acta, 66, 1335 (1983)]. (b) Thecase of sulfuric acid groupcanbeprepared by reacting the hydroxyl group of tyrosine withpyridine-sulfur trioxide complex. Further, (c) the case in which D isthe protected guanido group can be prepared by hydrogenizing the generalformula (XXV) having nitro group, converting to amino group, and then byreacting with 1H-pyrazole-N,N′-bisbenzyloxycarbonyl-1-carboxamidine.

(b) As the reaction solvent of the sulfurization, there is no specialrestriction if it does not hinder the proceeding of the reaction anddissolve the starting material, though, examples cited are alcohols,DMF, DMAc, ethyl acetate, diethyl ether, pyridine or the like,preferably DMF, DMAc or pyridine. The reaction temperature is usuallyfrom −10 to 40° C., preferably from −5 to 20° C. The reaction time isdifferent depending on solvent, reaction temperature, etc., though,usually from 30 min. to 24 hours, preferably from 1 to 4 hours.

(c) In the case of the conversion into a protected guanido group, as thehydrogenation catalyst hydrogenizing nitro group can be used catalystsuch as palladium-carbon, platinum or the like, preferablypalladium-carbon. As the reaction solvent can be used an inactiveorganic-solvent such as alcohols (methanol, ethanol, etc.), DMF, DMAc oracetic acid, which are not catalytic poison, or water, preferablymethanol. The reaction temperature is usually from 0 to 50° C.,preferably from 10 to 30° C. The reaction time is different depending ona starting material, solvent, reaction temperature, etc., though,usually from 1 to 24 hours, preferably from 1 to 6 hours. Subsequently,for reacting with 1H-pyrazole-N,N′-bis-benzyloxycarbonyl-1-carboxamidineas the reaction solvent is used an inactive organic-solvent such asn-hexane, benzene, toluene, DMF, DMAc, dichloromethane, chloroform, orthe like, though, preferably dichloromethane. The reaction temperatureis usually from −10 to 50° C., preferably from 0 to 30° C. The reactiontime is different depending on a starting material, solvent, reactiontemperature, etc., though, usually from 10 min. to 12 hours, preferablyfrom 30 min. to 6 hours.

The general formula (XXIII) can be prepared by introducing a protectinggroup such as Boc, Z or substituted Z groups or the like to the aminogroup of the general formula (XXII) by a conventional method.

For example, the case of Z group can be introduced by reactingchlorobenzyl formate in the presence of a base used conventionally(sodium carbonate, potassium carbonate, sodium bicarbonate, sodiumhydroxide, potassium hydroxide).

As the reaction solvent can be cited dioxane, acetone, water or a mixedsolvent thereof. The reaction temperature is usually from −20 to 30° C.,preferably from −5 to 5° C. The reaction time is usually from 2 to 24hours, preferably from 6 to 15 hours.

The general formula (XXV) can be prepared by reacting the generalformula (XXIII) with the amine of the general formula (XXIV) accordingto a conventional coupling technology (R³ has the same meaning describedabove, though, preferably hydrogen atom. R⁴ has the same meaningdescribed above, though, preferably a (C₁-C₄) alkyl group or an arylgroup which can be substituted, more preferably methyl, phenyl,p-methoxyphenyl or 1-naphthyl.).

As the condensation agent can be used DCC, EDC.HCl,N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ),1-hydroxybenzotriazole (HOBt) derivative,N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) derivative,N-hydroxysuccinimide (HOSu) derivative, iso-butyloxycarbonyl chloride,monoalkylcarbonate derivative produced by reacting with ethyloxycarbonylchloride, diphenylphosphoryl azide (DPPA) or the like, though,preferably EDC.HCl. As the reaction solvent, there is no specialrestriction if it does not hinder the proceeding of the reaction anddissolve the starting material, though, examples cited are DMF, DMAc,ethyl acetate, diethyl ether, dichloromethane, chloroform, dioxane orthe like, preferably DMF.

The reaction temperatureisusually from −20 to 20° C., preferably from−15 to 0° C.

The reaction time is usually from 1 to 24 hours, preferably from 2 to 15hours.

The intermediate (III) can be prepared by removing the amino-protectinggroup of the general formula (XXV). A conventional method is appliedcorresponding to a removing method and its type. For example, in thecase that the protecting group is Z group, the hydrogenation method is apreferable one. As a hydrogenation catalyst can be used catalysts suchas palladium-carbon, platinum or the like as a hydrogenation catalyst,though, preferably palladium-carbon. As the reaction solvent can be usedan inactive organic-solvent such as alcohols (methanol, ethanol, etc.),DMF, DMAc or acetic acid, which are not catalytic poison, or water,preferably methanol. The reaction temperature is usually from 0 to 50°C., preferably from 10 to 30° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 1 to 24 hours,preferably from 1 to 6 hours.

In the following, the preparation process of the preparationintermediates (IV), (V), (VI), (VII) and the target compounds (I) isexplained.

Synthetic routes of intermediates (IV), (V), (VI), (VII) and the targetcompounds (I)

(In the above synthetic route, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and Dhave the same meaning described above.)

The compound of the general formula (IV) can be prepared by reacting thepreparation intermediate (II) with the preparation intermediate (III)according to a conventional coupling technology (R⁷ has the same meaningdescribed above, though, preferably tert-butyl, benzyl, substitutedbenzyl, phenacyl or 2,2,2-trichloroethyl groups, especially tert-butylor benzyl groups.).

As the condensation agent used in this reaction can be cited DCC,EDC.HCl, EEDQ, HOBt derivative, HONB derivative, HOSu derivative,iso-butyloxycarbonyl chloride, monoalkylcarbonate derivative produced byreacting with ethyloxycarbonyl chloride, DPPA or the like, though,preferably EDC.HCl.

As the reaction solvent, there is no special restriction if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, examples cited are DMF, DMAc, ethyl acetate, diethylether, dichloromethane, chloroform, dioxane or the like.

The reaction temperature is usually from −20 to 20° C., preferably from−15 to 0° C. The reaction time is usually from 1 to 24 hours, preferablyfrom 2 to 15 hours.

The compound of the general formula (XXVI) can be prepared by thedeesterification of the compound of the general formula (IV). Forexample, in case of the tert-butyl ester, the deesterification can becarried out by the treatment with trifluoroacetic acid (TFA) or asolution, whereby hydrogen chloride is dissolved in ethyl acetate ordioxane.

The reaction temperature is usually from −10 to 20° C., preferably from−5 to 50° C. The reaction time is different depending on astartingmaterial, acid, reaction temperature, etc., though, usually from1 to 24 hours, preferably from 1 to 15 hours.

The compound of the general formula (XXVII) can be prepared bydeesterifying the compound of the formula (XI). In order to obtain thecompound of the general formula (XXVII), it is preferable that R⁷ and R⁸are benzyl groups and R⁹ is tert-butyl group.

For example, the deesterification of tert-butyl ester group can becarried out by the treatment with TFA or a solution, whereby hydrogenchloride is dissolved in ethyl acetate or dioxane.

The reaction temperature is usually from −10 to 20° C., preferably from−5 to 5° C. The reaction time is different depending on a startingmaterial, acid, reaction temperature, etc., though, usually from 1 to 24hours, preferably from 1 to 15 hours.

The compound of the general formula (XXVIII) can be prepared by reactingthe compound of the formula (XXIII) with the preparation intermediate(III) according to a conventional coupling technology.

As the condensation agent used can be cited DCC, EDC.HCl , EEDQ, HOBtderivative, HONB derivative, HOSu derivative, iso-butyloxycarbonylchloride, monoalkylcarbonate derivative produced by reacting withethyloxycarbonyl chloride, DPPA or the like, though, preferably EDC.HCl.

As the reaction solvent, there is no special restriction if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, examples cited are DMF, DMAc, ethyl acetate, diethylether, dichloromethane, chloroform, dioxane or the like, preferably DMF.

The reaction temperature is usually from −20 to 20° C., preferably from−15 to 0° C. The reaction time is usually from 1 to 24 hours, preferablyfrom 2 to 15 hours.

The general formula (XXIX) can be prepared by deesterifying two estergroups of the formula (XXVIII).

In order to obtain the compound of the general formula (XXIX), it ispreferable that R⁷ and R⁸ of the formula (XXVIII) are benzyl groups. Inthe case that R⁷ and R⁸ are benzyl groups, the debenzylation can becarried out easily by a hydrogenation method. As a hydrogenationcatalyst can be used catalysts such as palladium-carbon, platinum or thelike as a hydrogenation catalyst, though, preferably palladium-carbon.

As the reaction solvent can be used an inactive organic-solvent such asalcohols (methanol, ethanol, etc.), DMF, DMAc or acetic acid, which arenot catalytic poison, or water, preferably methanol.

The reaction temperature is usually from 0 to 50° C., preferably from 10to 30° C.

The reaction time is different depending on a starting material,solvent, reaction temperature, etc., though, usually from 1 to 24 hours,preferably from 1 to 6 hours.

The compound of the synthetic intermediate (V) can be prepared byreacting the Schiff base, resulting from formaline and a secondary aminesuch as piperidine, diethylamine, morpholine or the like (preferablypiperidine), with the general formula (XXIX), followed by thedecarboxylation and the simultaneous double-bond formation.

As the reaction solvent, there is no special restriction if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, examples cited are alcohols, DMF, DMAc or the like,preferably methanol or ethanol.

The reaction temperature is usually from 0 to 150° C., preferably from10 to 100° C. The reaction time is different depending on a startingmaterial, solvent, reaction temperature, etc., though, usually from 1 to24 hours, preferably from 1 to 15 hours.

The general formula (VI) can be prepared by reacting the hydroxylamine,in which carboxyl and hydroxyl groups in the preparation intermediate(V) are protected, using a conventional coupling technology (as ahydroxyl-protecting group can be cited nonsubstituted or substitutedbenzyl, trialkylsilyl, tert-butyldiphenylsilyl, tetrahydropyranyl ortert-butyl groups, or the like, preferably benzyl group.).

As the condensation agent used can be cited DCC, EDC.HCl , EEDQ, HOBtderivative, HONB derivative, HOSu derivative, iso-butyloxycarbonylchloride, monoalkylcarbonate derivative produced by reacting withethyloxycarbonyl chloride, DPPA or the like, though, preferably EDC.HCl.

As the reaction solvent, there is no special restriction if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, examples cited are DMF, DMAc, ethyl acetate, diethylether, dichloromethane, chloroform, dioxane or the like, preferably DMF.

The reaction temperature is usually from −20 to 20° C., preferably from−15 to 0° C. The reaction time is usually from 1 to 72 hours, preferablyfrom 2 to 48 hours.

The general formula (VII) can be prepared by (a) reacting thepreparation intermediate (III) and the formula (XXX) by a conventionalcoupling technology, or (b) reacting the carboxylic acid of the formula(XXVI) with the hydroxylamine, in which the hydroxyl group is protected,by a conventional coupling technology (As a hydroxyl-protecting groupcan be cited nonsubstituted or substituted benzyl, trialkylsilyl,tert-butyldiphenylsilyl, tetrahydropyranyl or tert-butyl groups, or thelike, preferably benzyl group.).

(a), (b) As the condensation agent used can be cited DCC, EDC.HCl, EEDQ,HOBt derivative, HONB derivative, HOSu derivative, iso-butyloxycarbonylchloride, monoalkylcarbonate derivative produced by reacting withethyloxycarbonyl chloride, DPPA or the like, though, preferably EDC.HCl.

As the reaction solvent, there is no special restriction if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, examples cited are DMF, DMAc, ethyl acetate, diethylether, dichloromethane, chloroform, dioxane or the like, preferably DMF.The reaction temperature is usually from −20 to 20° C., preferably from−15 to 0° C. The reaction time is usually from 1 to 72 hours, preferablyfrom 2 to 48 hours.

The general formula (XXX) can be prepared by the addition reaction ofthe hydroxylamine, in which the hydroxyl group is protected, to the acidanhydride (XXI).

As the reaction solvent, there is no special restriction if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, examples cited are DMF, DMAc, ethyl acetate, diethylether, benzene, toluene, dichloromethane, chloroform, dioxane or thelike, preferably DMF. The reaction temperature is usually from −20 to20° C., preferably from −10 to 10° C. The reaction time is usually from1 to 24 hours, preferably from 2 to 15 hours.

The compound of the general formula (I) can be prepared by (a) removingthe protecting group of the general formula (VII), (b) removing thehydroxyl-protecting group after adding a thiol derivative to the generalformula (VI), (c) reacting the carboxylic acid of the general formula(XXVI) with hydroxylamine or its salt by a conventional couplingtechnology or (d) the aminolysis of the ester of the general formula(IV) by hydroxylamine.

(a) As a method to remove the hydroxyl-protecting group, in the casethat the protecting group is benzyl group, whereby it can be removed bya conventional hydrogenation method, however, in the case that sulfuratom is present in the molecule, whereby hydrofluoric acid is preferablyused for the debenzylation.

In case of removing benzyl group by the hydrogenation method, as ahydrogenation catalyst can be used catalysts such as palladium-carbon,platinum or the like as a hydrogenation catalyst, though, preferablypalladium-carbon.

As the reaction solvent can be used an inactive organic-solvent such asalcohol (methanol, ethanol, etc.), DMF, DMAc or acetic acid, which arenot catalytic poison, or water, preferably methanol or acetic acid.

The reaction temperature is usually from 0 to 50° C., preferably from 10to 30° C.

The reaction time is different depending on the starting material,solvent, reaction temperature, etc., though, usually from 1 to 24 hours,preferably from 1 to 6 hours.

In case of treating with hydrofluoric acid, as a scavenger is addedanisole, dimethyl sulfide, ethanedithliol, methionine or the like.

The reaction temperature is usually from −40 to 30° C., preferably from−5 to 5° C. The reaction time is different depending on a startingmaterial, reaction temperature, etc., though, usually from 10 min. to 3hours, preferably from 30 min. to 1 hour.

(b) In the method in which a thiol derivative is added to the generalformula (VI), there is no special restriction for the reaction solventif it does not hinder the proceeding of the reaction and dissolve thestarting material, though, examples cited are alcohols, DMF, DMAc,dichloromethane, chloroform or the like, preferably methanol. Further,the reaction can be done without solvent.

The reaction temperature is usually from 0 to 100° C., preferably from 0to 60° C. The reaction time is largely different depending on thestarting material, solvent, reaction temperature, etc., though, usuallyfrom 10 hours to 1 month, preferably from 10 hours to 2 weeks.

Next, in the method to remove a hydroxyl-protecting group is applied themethod to treat with hydrofluoric acid when the protecting group isbenzyl group. In case of treating with hydrof luoric acid, as ascavenger is added anisole, dimethyl sulfide, ethanedithiol, methionineor the like.

The reaction temperature is usually from −40 to 30° C., preferably from−5 to 5° C. The reaction time is different depending on a startingmaterial, reaction temperature, etc., though, usually from 10 min. to 3hours, preferably from 30 min. to 1 hour.

(c) In the method that the carboxylic acid of the formula (XXVI) andhydroxylamine and salts thereof are reacted according to a conventionalcoupling technology, as the condensation agent used can be cited DCC,EDC.HCl, EEDQ, HOBt derivative, HONB derivative, HOSu derivative,iso-butyloxycarbonyl chloride, monoalkylcarbonate derivative produced byreacting with ethyloxycarbonyl chloride, DPPA or the like, though,preferably EDC.HCl.

As the above reaction solvent, there is no special restriction if itdoes not hinder the proceeding of the reaction and dissolve the startingmaterial, though, examples cited are DMF, DMAc, ethyl acetate, diethylether, dichloromethane, chloroform, dioxane or the like, preferably DMF.The reaction temperature is usually from −20 to 20° C., preferably from−15 to 0° C. The reaction time is usually from 1 to 24 hours, preferablyfrom 2 to 15 hours.

(d) In the aminolysis of the ester of the formula (IV) by hydroxylamine,there is no special restriction for the reaction solvent if it does nothinder the proceeding of the reaction and dissolve the startingmaterial, though, examples used are alcohols (methanol, ethanol, etc.),DMF, DMAc, ethyl acetate, diethyl ether, dichloromethane, chloroform,dioxane or the like, preferably alcohols.

The reaction temperature is usually from −30 to 200° C., preferably from−10 to 30° C. The reaction time is different depending on the startingmaterial, reaction temperature, etc., though, usually from 10 min. to 24hours, preferably from 30 min. to 10 hours. Further, if the reactiondoes not proceed easily, it is possible to let it proceed by thepressurization.

Diseases concerned with a tissue destruction to which the compoundsobtained by the invention are applied, mean chronic rheumatoidarthritis, osteoarthritis, periodental disease, corneal ulcer, variouskinds of bullosis (epidermolisis bullosahereditaria, epidermolisisbullosa acquisita, porphylia cutanea tarda, bullos penphigoid, pemphigusvulgaris), intractable skin ulcer (bedsore, skin ulcer in radiotherapy,skin ulcer in diabetes mellitus, skin ulcer in arterioscleroticobliteration), wound (external injury or burn), osteoporosis, cancermetasis and the like. The compounds obtained by the invention are, forexample,

N-[4-(N-Hydroxyamino)-2(R orS)-isobutylsuccinyl]-O-sulfo-L-tyrosine-N-methylamide, monosodium salt;

N-[4-(N-Hydroxyamino)-2(RS)-isobutylsuccinyl]-L-4′-sulfophenylalanine-N-methylamide,monosodium salt;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-methylamide,monosodium salt;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,monosodium salt;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(thienylthiomethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,monosodium salt;

N-[4-(N-Hydroxyamino)-2(R)-n-propyloxymethlene-3(RS)-isopropylthiomethylenesuccinyl]-L-4′-sulfophenylalanine-N-methylamide,monosodium salt;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-p-methoxyphenylamide,monosodium salt;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-guanidophenylalanine-N-methylamide monoacetate;

N-[4-(N-Hydroxyamino)-2(R)-isobutylsuccinyl]-O-phospho-L-tyrosine-N-methylamide,disodium salt;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-acetimidoyliminomethylenephenylalanine-N-methylamidemonoacetate;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-propionimidoyliminomethylenephenylalanine-N-methylamidemonoacetate;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-benzimidoyliminomethylenephenylalanine-N-methylamidemonoacetate;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-acetamidomethylenephenylalanine-N-methylamide;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-guanidomethylenephenylalanine-N-methylamidemonoacetate;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-meta-tyrosine-N-methylamide,monosodium salt;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-hydroxysuccinyl]-L-4′-guanidophenylalanine-N-methylamidemonoacetate;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-amidinophenylalanine-N-methylamidemonoacetate;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-1-naphthylamide,monosodium salt;

N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-guanidophenylalanine-N-phenylamidemonoacetate, etc.

These compounds have excellent inhibitory activities for MMPs and/orTNF-α converting enzyme, and additionally, the water-solubility thereofwas conspicuously increased compared with the compounds knownpreviously. Consequently, these compounds are not only easy for theapplication to the diseases concerned with the tissue destruction, butalso are useful compounds by which the treatment and/or a preventiveeffect can be expected.

In the following are explained assay methods for biological activitiesof the compounds according to the inventions, their pharmaceuticalpreparations, dose, etc.

Inhibitory activities of the compounds represented by the generalformula (I) according to the invention for collagenase(metalloproteinase concerned with a tissue destruction) derived fromhuman fibroblast were tested by the method of Y. Murawaki et al.,[Journal of Hepatology, 18, p. 328-334 (1993)], and their inhibitoryactivities for stromelysin derived from human fibroblast were measuredby the method of S. S. Twining et al., [Anal. Biochem., 143, p. 30(1984)]. The test results are described below as test examples.

The compounds of the general formula (I) can be administered by any ofdosage forms such as oral, topical, parenteral administration or thelike. As those corresponding to these dosage forms, they are provided informs such as tablets, capsules, powders, granules, fluids or gelpreparation. In the tablets or the capsules for oral administration, thedosage unit is in a written form, and they can be let include usualadditives shown below by using a conventional method. The tablets can becoated according to the well-known method in a conventionalpharmaceutical operation.

(1) Binders such as liquid glucose, acacia, gelatin, methyl cellulose,povidone or the like;

(2) Excipients such as lactose, starch, calcium phosphate, sorbitol orthe like;

(3) Lubricants, for example, such as magnesium stearate, talc,polyethylene glycol or the like;

(4) Disintegraters such as starch and wetting agents such as sodiumlaurylsulfate.

Oral liquid preparations are composed in forms, for example, such assuspensions, solutions, emulsions, syrups or elixirs, containing wateror oil portions, or are provided as a dry product to be converted to aliquid state by adding water or the other excipients.

These liquid preparations can include suspension agents , for example,such as tragacanth, polyvinyl alcohol, methyl cellulose or gelatin;emulsifiers, for example, such as lecithin, sorbitan ester or acacia;plasticizers, for example, such as glycerin, propylene glycol,antimicrobial preservatives or sterilizing preservatives, for example,such as methylparaben or propylparaben, or usual additives like sorbicacid and, if necessary, also usual sweeteners or colorings.

The dosage unit of oral administration is about 0.1-500 mg, andpreferably, it contains about 1-250 mg of a compound of the generalformula (I) The appropriate daily dose is largely different depending onthe state of a patient, though, appropriate doses of a compound of thegeneral formula (I) are 0.001-300 mg/Kg body weight, especially 0.01-100mg/Kg body weight.

Pharmaceutical substances for topical application to skin can beprepared as solutions or suspension liquids by sterile water ornonaqueous excipients. As additives are cited, for example, buffersubstances such as citric acid, sodium citrate (dihydrate or anhydride)or edetate salts; preservatives containing antiseptic agents orantimicrobial agents such as phenylmercuric nitrate, benzalkoniumchloride or chlorhexidine, and tackifiers like hydroxypropyl methylcellulose. The dosage for topical administration is different dependingon the size of a part treated, though, the dosage unit for eye (one eye)is in a range of 0.1-100 mg of a compound of the general formula (I). Anactive ingredient can be administered parenterally by using sterilevehicles.

Pharmaceutical substances can be suspended or dissolved in excipientsaccording to the excipients used and the concentration thereof.Auxiliaries such as local anesthetic agents, preservatives and buffersubstances are soluble in exicipients. Concerning the use for thetreatment of arthritis such as osteoarthritis and chronic rheumatoidarthritis, compounds of the invention can be administered orally or byinjecting into diseased joint intraarticularly. The daily dosage formammal of body weight, 70 Kg, is in a range of 0.1-10 g of a compound ofthe general formula (I).

In the following, the invention will be explained in more detail byshowing examples, test examples and preparations' example, but theinvention is not limited thereby in any way. The abbreviations describedbelow are used in the examples as they are understood to mean thefollowings. Further, specific rotation was measured at 25° C.

DMF; N,N-dimethylformamide, DMSO; dimethylsufoxide, EDC;1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, HOBT;1-hydroxybenzotriazole, TEA; triethylamine, THF; tetrahydrofuran.

EXAMPLE 1 N-[4-(N-Hydroxyamino)-2(R orS)-isobutylsuccinyl]-O-sulfo-L-tyrosine-N-methylamide, Monosodium Salt

(a) Benzyl 2-isobutyl-3-tert-butoxycarbonylpropenoate

To a suspension of 60% NaH (3.60 g, 90.0 mmol) in anhydrous THF (100ml), which was stirred under ice-cooling and nitrogen atmosphere, wasdropped (tert-butoxycarbonylmethylene) triphenylphosphrane (22.8 g, 90.4mmol). After stirring under ice-cooling for 30 min., the mixture wasadded with benzyl 4-methyl-2-oxopentanoate (13.0 g, 59.0 mmol) andstirred at room temperature overnight. The reaction mixture was addedwith Et₂O (300 ml) and washed with water and then with a saturatedaqueous solution of NaCl two times respectively. The organic layer wasdried over anhydrous MgSO₄, evaporated under reduced pressure andpurified by column chromatography (silica gel; 500 g, elution with themixed solvent; n-hexane: AcOEt=20:1), giving the title compound (15.3 g,81%) of a colorless oil.

Rf value; 0.37 (AcOEt:n-hexane=1:10).

¹H-NMR(CDCl₃) δ ppm; 0.88 (6H, d, J=6.5 Hz, CH(CH₃)₂ ), 1.43 (9H, s,(CH₃)₃ C), 1.5-1.9 (1H, m, (CH₃)₂ CH—CH₂), 2.22 (2H, bd, J=7.2 Hz,(CH₃)₂CH—CH₂ ), 5.22 (2H, s, O—CH₂ ), 7.37 (5H, s, aromatic-H).

(b) 2(RS)-Isobutyl-3-tert-butoxycarbonylpropanoic Acid

The compound (15.0 g, 47.1 mmol) of Example 1-a was dissolved in AcOH(200 ml). The solution was added with 10% Pd-C (50%, 10.0 g) and thenstirred vigorously in a stream of hydrogen at room temperature for 5hours. After the catalyst was filtered off, the solvent was removedunder reduced pressure to give the title compound (10.8 g, inquantitative yield) of a colorless oil.

¹H-NMR(CDCl₃) δ ppm; 0.7-1.0 (6H, m, CH(CH₃)₂ ), 1.1-1.8 (12H, m, (CH₃)₂CH—CH_(2,) +C(CH₃)₃ ), 2.44 (2H, dd, J=10, 8 Hz, CH₂ —COOC(CH₃)₃), 2.8(1H, m, CH—CO), 8.4 (1H, brs, COOH).

(c) N-[4-tert-Butoxy-2(R orS)-isobutylsuccinyl]-L-tyrosine-N-methylamide

The compound (4.60 g, 20.0 mmol) of Example 1-b,L-tyrosine-N-methylamide hydrochloride (4.90 g, 21.4 mmol), HOBT (5.40g, 40.0 mmol) were dissolved in DMF (30 ml) and CH₂Cl₂ (10 ml), and thesolution was added with TEA (3.10 ml, 22.1 mmol) and EDC (5.00 g, 26.1mmol) under stirring at −15° C. The mixture was stirred at −15° C. for 1hour and further at room temperature overnight and evaporated underreduced pressure. The residue was added with AcOEt (300 ml) and washedwith a saturated aqueous solution of NaCl, 1N-HCl , a saturated aqueoussolution of NaHCO₃ and then the saturated aqueous solution of NaCl twotimes respectively. The organic layer was dried over anhydrous MgSO₄ andevaporated under reduced pressure. The residue was added with Et₂O (150ml), giving crystals (2.55 g, 32%, one isomer of the title compounds:designating isomer A). The filtrate was evaporated under reducedpressure and purified by column chromatography separation (silica gel;250 g, elution with the mixed solvent; n-hexane: AcOEt1:2), giving thetitle compound (3.70 g, 46%, the other isomer of the title compounds:designating isomer B) of a pale yellow solid.

Isomer A

m.p.; 211° C., specific rotation[α]_(D)=−18.2° (c=1.0, MeOH), Rf value;0.63 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(CDCl₃) δ ppm; 0.75 (6H, d, J=4.6 Hz, CH(CH₃)₂ ), 0.8-1.7 (12H,s+m, (CH₃)₂ CH—CH_(2,) +C(CH₃)₃ ), 2.0-3.5 (8H, d+m, J=4.6 Hz, N—CH₃+CO—CH₂CH—CO+CH₂ —C₆H₄), 4.7 (1H, m, NH—CH—CO), 6.6, 7.4 (1H×2, m,NH×2), 6.87 (4H, AA′BB′, aromatic-H).

Isomer B

m.p.; 62° C., specific rotation[α]_(D)=−4.0° (c=1.1, MeOH), Rf value;0.63 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(CDCl₃) δ ppm; 0.85 (6H, m, CH(CH₃)₂ ), 1.0-1.8 (12H, s+m, (CH₃)₂CH—CH_(2,) +C(CH₃)₃ ), 2.3-2.8 (7H, d+m, J=4.6 Hz, N—CH₃ +CH₂ COO+CH₂—C₆H₄), 3.0 (1H, m, CH—CO), 4.5 (1H, m, NH—CH—CO), 5.9, 6.3 (1H×2, m,NH×2), 6.90 (4H, AA′BB′, aromatic-H).

(d) N-[4-(N-Hydroxy)-2(R orS)-isobutylsuccinyl]-L-tyrosine-N-methylamide

The compound (isomer B, 3.50 g, 8.60 mmol) of Example 1-c was added withice-cooled 95% aqueous trifluoroacetic acid (10 ml). The reactionmixture was stirred at 5° C. overnight and evaporated under reducedpressure. The residue was added with Et₂O. After stirring at roomtemperature for 1 hour, the crystallized solid was filtered to give thetitle compound (isomer B, 1.85 g, 61%) of a white solid upon drying.

Also from the compound (isomer A, 2.44 g, 6.00 mmol) of Example 1-c, thetitle compound (isomer A, 2.00 g, 95%) of a white solid was obtained bythe similar procedure.

Isomer B

m.p.; 214° C., specific rotation[α]_(D)=−0.2° (c=1.0, MeOH), Rf value;0.17 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(MeOH-d₄) δ ppm; 0.82 (6H, m, CH(CH₃)₂ ), 1.0-1.6 (3H, m, (CH₃)₂CH—CH_(2,) ), 2.1-3.5 (m, N—CH₃ +CO—CH₂CH—CO+CH₂ —C₆H₄), 4.43 (1H, m,NH—CH—CO), 6.85 (4H, AA′BB′, aromatic-H).

Isomer A

m.p.; 102° C., specific rotation[α]_(D)=−21.7° (c=1.1, MeOH), Rf value;0.12 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.5 (9H, m, (CH₃)₂CH—CH₂ ), 2.0-3.7 (m, N—CH₃+CO—CH₂CH—CO+CH—CO+CH₂ —C₆H₄), 4.65 (1H, dd, J=5.3, 9.3 Hz, NH—CH—CO),6.88 (4H, AA′BB′, aromatic-H).

(e) N-[4-(N-Benzyloxyamino)-2(R orS)-isobutylsuccinyl]-L-tyrosine-N-methylamide

The compound (isomer B, 3.51 g, 10.0 mmol) of Example 1-d,O-benzylhydroxylamine hydrochloride (3.20 g, 20.0 mmol) and HOBT (2.70g, 20.0 mmol) were suspended in DMF (20 ml) and added with TEA (2.80 ml,20.0 mmol) and EDC (3.80 g, 20.0 mmol) at −15° C. under stirring. Thesuspension was stirred at −15° C. for 1 hour and further at roomtemperature overnight and evaporated under reduced pressure. The residuewas added with AcOEt (250 ml) and washed quickly with a saturatedaqueous solution of NaCl, 1N-HCl, a saturated aqueous solution of NaHCO₃and then the saturated aqueous solution of NaCl two times respectively.The organic layer was dried over anhydrous MgSO₄ and evaporated underreduced pressure. The residue was added with Et₂O and stirred at roomtemperature for 1 hour. The crystallized solid was filtered to give thetitle compound (isomer B, 3.90 g, 86%) of a white solid upon drying in adesiccator under reduced pressure.

Also from the compound (isomer A, 2.44 g, 6.95 mmol) of Example 1-d, thetitle compound (isomer A, 2.40 g, 84%) of a white solid was obtained bythe similar procedure.

Isomer B

m.p.; 198° C., specific rotation[α]_(D)=−18.8° (c=1.1, MeOH), Rf value;0.31 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(MeOH-d₄+CDCl₃) δ ppm; 0.6-1.7 (9H, m, (CH₃)₂CH—CH₂ ), 2.08 (2H,bd, J=4.6 Hz, CH₂ CO), 2.5-3.1 (6H, m, N—CH₃ +CH—CO+CH₂ —C₆H₄), 4.45(1H, m, NH—CH—CO), 4.83 (2H, s, O—CH₂ ), 6.87 (4H, AA′BB′,tyrosin(aromatic-H)), 7.31 (5H, s, aromatic-H).

Isomer A

m.p.; 97° C., specific rotation[α]_(D)=−20.0° (c=1.0, MeOH), Rf value;0.31 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(MeOH-d₄+CDCl₃) δ ppm; 0.5-1.7 (9H, m, (CH₃)₂CH—CH₂ ), 1.9-3.5(8H, m, N—CH₃ +CO—CH₂CH—CO+CH₂ —C₆H₄), 4.45-4.95 (3H, m, NH—CH—CO+O—CH₂), 6.9 (4H, AA′BB′, tyrosine (aromatic-H)), 1.35 (5H, s, aromatic-H).

(f) N-[4-(N-Benzyloxyamino)-2(R orS)-isobutylsuccinyl]-O-sulfo-L-tyrosine-N-methylamide, Monosodium Salt

The compound (isomer B, 1.50 g, 3.30 mmol) of Example 1-e was suspendedin DMF (3.3 ml). The suspension was added with sulfur trioxide pyridinecomplex (1.60 g, 9.90 mmol) and stirred at room temperature for 1.5hours. The reaction liquid was added with 1N-NaHCO₃ (80 ml), stirred atroom temperature for 40 min. and then purified by a reverse-phase columnchromatography (Fuji Silysia Chemical Ltd., Chromatorex ODS DM-1020T; 75g, elution with a MeOH-water solution containing 0-50% MeOH) to give thetitle compound (isomer B, 1.60 g, 87%) of white amorphous powder uponfreeze-drying.

Also from the compound (isomer A, 1.80 g, 3.95 mmol) of Example 1-e, thetitle compound (isomer A, 1.85 g, 84%) of white amorphous powder wasobtained by the similar procedure.

Isomer B

Specific rotation[α]_(D)=−14.7° (c=0.4, MeOH), Rf value; 0.87(CHCl₃:MeOH:AcOH=5:2:1), 0.81 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.5 (9H, m, (CH₃)₂CH—CH₂ ), 1.9-3.0 (8H, m,N—CH₃ +CO—CH₂CH—CO+CH₂ —C₆H₄), 4.50 (m, NH—CH—CO), 7.20 (4H, s, tyrosine(aromatic-H)), 7.35 (5H, m, aromatic-H).

Isomer A

Specific rotation[α]_(D)=−15.3° (c=1.0, MeOH), Rf value; 0.87(CHCl₃:MeOH:AcOH=5:2:1), 0.81 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.82 (6H, m, (CH₃)₂ CH—CH₂), 0.9-1.6 (3H, m,(CH₃)₂ CH—CH₂ ), 2.0-3.0 (8H, m, CO—CH₂CH—CO+N—CH₃ +CH₂ —C₆H₄), 4.50(1H, m, NH—CH—CO), 7.20 (4H, brs, tyrosine (aromatic-H)), 7.37 (5H, m,aromatic-H).

(g) N-[4-(N-Hydroxyamino)-2(R orS)-isobutylsuccinyl]-O-sulfo-L-tyrosine-N-methylamide, Monosodium Salt

The compound (isomer B, 1.60 g, 2.87 mmol) of Example 1-f was dissolvedin MeOH (100 ml). The solution was added with 10% Pd-C (50% wet, 0.50 g)and then stirred vigorously in a stream of hydrogen at room temperaturefor 1 hour. After the catalyst was filtered off, MeOH of the filtratewas removed under reduced pressure. The residue was added with water (50ml) and lyophilized to give the title compound (isomer B, 1.22 g, 90%)of white amorphous powder.

Also from the compound (isomer A, 0.50 g, 0.90 mmol) of Example 1-f, thetitle compound (isomer A, 0.39 g, 92%) of white amorphous powder wasobtained by the similar procedure.

Isomer B

Specific rotation[α]_(D)=−7.5° (c=0.51, MeOH), Rf value; 0.58(CHCl₃:MeOH:AcOH=5:2:1), 0.61 (n-BuOH:AcOH:water=4:1:1). Analyticalvalue calculated for C₁₈H₂₆N₃O₈SNa. Theoretical value:C, 46.25; H, 5.61;N, 8.99. Found:C, 46.0; H, 5.83; N, 8.77.

¹H-NMR(MeOH-d₄) δ ppm; 0.83 (6H, m, (CH₃)₂ CH—CH₂), 1.0-2.0 (3H, m,(CH₃)₂ CH—CH₂ ), 2.08 (2H, m, CH₂ CO), 2.5-3.1 (6H, m, CH—CO+N—CH₃ +CH₂—C₆H₄), 4.50 (1H, m, NH—CH—CO), 7.70 (4H, s, aromatic-H).

Isomer A

Specific rotations[α]_(D)=−8.1° (c=1.0, MeOH), Rf value; 0.55(CHCl₃:MeOH:AcOH=5:2:1), 0.60 (n-BuOH:AcOH:water=4:1:1). Analyticalvalue calculated for C₁₈H₂₆N₃O₈SNaH₂O. Theoretical value:C, 44.53; H,5.81; N, 8.66. Found:C, 44.41; H, 5.88; N, 8.65.

¹H-NMR(MeOH-d₄+CDCl₃) δ ppm; 0.6-1.7 (9H, m, (CH₃)₂CH—CH₂ ), 1.9-3.5(8H, m, N—CH₃ +CO—CH₂CH—CO+CH₂ —C₆H₄), 7.70 (4H, s, aromatic-H).

EXAMPLE 2N-[4-(N-Hydroxyamino)-2(RS)-isobutylsuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

(a) N-Benzyloxycarbonyl-L-4′-sulfophenylalanine, Monosodium Salt

L-4′-sulfophenylalanine [HELVETICA CHIMICA ACTA, 66, P. 1355 (1983),18.0 g, 73.4 mmol] and NaHCO₃ (24.7 g, 294 mmol) were dissolved in water(180 ml). The solution was added with benzyloxycarbonyl chloride (14.0ml, 88.1 mmol) and Et₂O (20 ml) under ice-cooling and stirring andstirred at room temperature for 5 hours. The reaction liquid was washedtwo times with AcOEt (200 ml), made pH 1-2 with 6N-HCl and extractedthree times with THF (200 ml) under the saturation of NaCl. The organiclayer was washed with a saturated aqueous NaCl solution (200 ml), driedover anhydrous MgSO₄ and evaporated under reduced pressure to give thetitle compound (29.5 g, in quantitative yield) as white crystals byevaporating the solvent under reduced pressure.

m.p.; 260° C. (dec.). Specific rotation[α]_(D)=−0.81° (c=1.0, MeOH). Rfvalue; 0.48 (CHCl₃:MeOH:AcOH=5:2:1), 0.50 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 2.8-3.3 (2H, m, C₆H₄—CH₂ ), 4.3-4.55 (1H, m,NH—CH—CO), 5.03 (2H, s, PhCH₂ ), 7.15-7.9 (10H, NH+aromatic-H).

(b) L-4′-Sulfophenylalanine-N-methylamide, Monosodium Salt

The compound (29.5 g, 73.5 mmol) of Example 2-a and HOBT (10.4 g, 77.2mmol) were dissolved in DMF (240 ml) and stirred at −15° C. The solutionwas dropped with an aqueous solution of methylamine hydrochloride (60ml, 5.96 g, 88.3 mmol), followed by the addition of EDC (16.2 g, 84.5mmol) and TEA (12.2 ml, 88.4 mmol) and stirred at 5° overnight. Thereaction liquid was concentrated under reduced pressure and thenpurified by column chromatography (silica gel; 1.5 Kg, elution with themixed solvent; CHCl₃:MeOH=9:1-4:1).

The obtained pale yellow oily product (36.5 g) was dissolved in MeOH(300 ml) and hydrogenated by catalyst at room temperature for 7 hours inthe presence of 10% Pd-C (50% wet, 2.3 g). After the catalyst wasfiltered off, the filtrate was evaporated under reduced pressure toremove MeOH and crystallized from MeOH-AcOEt, giving the title compound(16.2 g, 81%) as white crystals.

m.p.; ≧300° C. Specific rotation[α]_(D)=+43.3° (c=1.0, H₂O). Rf value;0.19 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(DMSO-d₆) δ ppm; 2.60 (NHCH₃ ), 2.7-3.0 (2H, m, C₆H₄ CH₂ ),3.4-3.7 (1H, m, H₂N—CH—CO), 3.9 (2H, brm, H₂N), 7.35 (4H, AA′BB′,aromatic-H), 7.9-8.2 (1H, m, CO—NH).

(c)N-[4-tert-Butoxy-2(RS)-isobutylsuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (8.32 g, 29.7 mmol) of Example 2-b, the compound (5.70 g,24.8 mmol) of Example 1-b, and HOBT (3.51 g, 26.0 mmol) were dissolvedin DMF (140 ml). The solution was added with EDC (5.69 g, 29.7 mmol) at−15° C. under stirring and stirred at room temperature overnight. Thereaction mixture was concentrated under reduced pressure, followed bydissolving the concentrate in THF (200 ml). The solution was washed with1N-HCl, a saturated aqueous solution of NaHCO₃ and then the saturatedaqueous solution of NaCl two times respectively. The organic layer wasdried over anhydrous MgSO₄, evaporated under reduced pressure to removethe solvent and purified by column chromatography (silica gel; 500 g,elution with the mixed solvent; CHCl₃:MeOH=9:1-4:1) to give the titlecompound (6.10 g, 50%) of a colorless oil.

m.p.; ≧300° C. Specific rotation[α]_(D)=−11.1° (c=1.0, MeOH). Rf value;0.56 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.6-0.95 (6H, m, CH(CH₃)₂ ), 0.9-1.8 (12H, m,C(CH₃)₃ +CH₂CH(CH₃)₂), 2.1-3.2 (7H, m, C₆H₄ CH₂ +(CH₃)₃CO₂C—CH₂ +NHCH₃), 3.4-3.7 (1H, m, CO—CHCH₂), 4.3 (2H, brm, 2×NH), 4.4-4.7 (1H, m,NH—CH—CO), 7.54 (4H, AA′BB′, aromatic-H).

(d)N-[4-(N-Benzyloxyamino)-2(RS)-isobutylsuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (0.29 g, 0.59 mmol) of Example 1-c was added to ice-cooled95% aqueous trifluoroacetic acid (3 ml) and stirred at 5° C. for 4hours. The reaction mixture was concentrated under reduced pressure. Theobtained colorless oil (0.25 g, 100%), O-benzylhydroxylaminehydrochloride (0.11 g, 0.71 mmol), HOBT (0.08 g, 0.62 mmol) and TEA(0.10 ml, 0.71 mmol) were dissolved in DMF (5 ml), added with EDC (0.13g, 0.68 mmol) at −15° C. under stirring and stirred at room temperatureovernight. The mixture was evaporated under reduced pressure to removethe solvent and purified by column chromatography (silica gel; 15 g,elution with the mixed solvent; CHCl₃:MeOH=9:1-4:1) to give the titlecompound (0.14 g, 43%) of a white solid.

m.p.; 111° C. Specific rotation[α]_(D)=−16.4° (c=1.0, MeOH). Rf value;0.62 (CHCl₃:MeOH:AcOH=5:2:1), 0.59 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.6 (9H, m, CH₂CH(CH₃)₂ ), 1.8-3.7 (8H, m,C₆H₄ CH₂ +CO—CH₂CH—CO+NHCH₃ ), 4.58 (1H, m, NH—CH—CO), 7.1-7.9 (9H,AA′BB′, aromatic-H).

(e)N-[4-(N-Hydroxyamino)-2(RS)-isobutylsuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (0.12 g, 0.22 mmol) of Example 1-d was dissolved in MeOH (5ml), added with 10% Pd-C (50% wet, 0. 12 g), and then stirred vigorouslyin a stream of hydrogen at room temperature for 5 hours. After thecatalyst was filtered off, MeOH of the filtrate was removed underreduced pressure. The residue was purified by column chromatography(silica gel; 10 g, elution with the mixed solvent; CHCl₃:MeOH=4:1-2:1)to give the title compound (0.06 g, 60%) of pale yellow crystals.

m.p.; 200-202° C., Specific rotation[α]_(D)=−12.2° (c=1.0, MeOH), Rfvalue; 0.33 (CHCl₃:MeOH:AcOH=5:2:1), 0.39 (n-BuOH:AcOH:water=4:1:1). Aanalytical value calculated for C₁₈H₂₆N₃O₇SNa. Theoretical value:C,47.89; H, 5.8; N, 9.31. Found:C, 47.66; H, 5.97; N, 9.15.

¹H-NMR(MeOH-d₄) δ ppm; 0.6-1.0 (6H, m, CH(CH₃)₂ ), 1.0-1.7 (3H, m,CH₂CH(CH₃)₂), 1.9-3.6 (8H, m, C₆H₄ CH₂ +CO—CH₂CH—CO+NHCH₃ ), 4.4-5.0(1H, m, NH—CH—CO), 4.85 (3H, brm, 3×NH), 7.53 (4H, AA′BB′, aromatic-H).

EXAMPLE 3 N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene) succinyl]-O-sulfo-L-tyrosine-N-methylamide,Monosodium Salt

(a) Benzyl 2(R)-bromo-4-metylpentanoate

2(R)-Bromo-4-metylpentanoic acid (28.5 g, 146 mmol), benzyl alcohol(18.1 ml, 175 mmol) and 4-dimethylaminopyridine (1.90 g, 14.6 mmol) weredissolved in CH₂Cl₂ (140 ml), and added with EDC (35.6 g, 175 mmol)under ice-cooling and stirring The mixture was stirred under ice-coolingfor 1 hour and further at room temperature overnight and washed forseparation with water, a saturated aqueous solution of NaHCO₃ and then asaturated aqueous solution of NaCl two times respectively. The organiclayer was dried over an hydrous MgSO₄, evaporated under reduced pressureand purified by column chromatography (silica gel; 700 g, elution withthe mixed solvent; n-hexane:AcOEt=40:1), giving the title compound (32.0g, 77%) of a colorless oil.

Specific rotation[α]_(D)=+31.8° (c=1.0, MeOH), Rf value; 0.48(AcOEt:n-hexane=1:5).

¹H-NMR(CDCl ³ ) δ ppm; 0.9 (6H, 2×d, J=6.5 Hz, CH(CH₃)₂ ), 1.67 (1H, m,(CH₃)₂ CH) 1.90 (2H, m, (CH₃)₂CH—CH₂ ), 4.30 (1H, t, J=7 Hz, —CH—Br),5.2 (2H, s, CH₂ —Ph), 7.32 (5H, s, aromatic-H).

(b) Dibenzyl 3(RS)-tert-butoxycarbonyl-2(R)-isobutylsuccinate

Benzyl tert-butyl malonate (24.9 g, 99.6 mmol) was dissolved in DMF (60ml) and added portion wise with potassium tert-butoxide (13.4 g, 120mmol) at 0° C. under stirring. The mixture was stirred at roomtemperature for 1 hour, then cooled to 0° C., and dropped in 1 hour withthe solution of the Example 3-a compound (28.4 g, 99.6 mmol) dissolvedin DMF (60 ml). After the reaction mixture was stirred at 5° C.overnight, it was added with AcOEt (2 l) and washed with a saturatedaqueous solution of NaCl, 1N-HCl, a saturated aqueous solution of NaHCO₃and then the saturated aqueous solution of NaCl two times respectively.The organic layer was dried over anhydrous MgSO₄, evaporated underreduced pressure and purified by column chromatography (silica gel; 750g, elution with the mixed solvent; n-hexane:AcOEt=20:1), giving thetitle compound (40.0 g, 89%) of a colorless oil.

Specific rotation[α]_(D)=+16.7° (c=1.0, MeOH), Rf value; 0.56(AcOEt:n-hexane=1:5).

¹H-NMR(CDCl₃) δ ppm; 0.82 (6H, 2x×d, J=10 Hz, CH(CH₃)₂ ), 1.15-1.8 (12H,2×s+m, (CH₃)₂ CH—CH₂ +C(CH₃)₃ ), 3.2 (1H, m, CH₂—CH—CO), 3.7 (1H, m,CO—CH—CO), 5.1 (4H, m, CH₂ —Ph×2), 7.32 (10H, s, aromatic-H).

(c) Dibenzyl 3(RS)-tert-butoxycarbonyl-3-cinnamyl-2(R)-isobutylsuccinate

The compound (9.49 g, 20.9 mmol) of Example 3-b was dissolved in DMF(100 ml), and added portion wise with 60% NaH (1.0 g, 25.1 mmol) at roomtemperature under stirring. The mixture was stirred at room temperaturefor 2 hours, cooled to 0° C., added portion wise with cinnamyl bromide(5.35 g, 27.2 mmol) and stirred at 5° C. for 15 hours. After removingthe solvent under reduced pressure, the residue was added with AcOEt(500 ml), and washed with a saturated aqueous solution of NaCl, 1N-HCl,a saturated aqueous solution of NaHCO₃ and then the saturated aqueoussolution of NaCl two times respectively. The organic layer was driedover anhydrous MgSO₄, evaporated under reduced pressure and purified bycolumn chromatography (silica gel; 700 g, elution with the mixedsolvent; n-hexane:AcOEt=20:1), giving the title compound (10.9 g, 91%)of a colorless oil.

Rf value; 0.34 (AcOEt:n-hexane=1:9).

¹H-NMR(CDCl₃) δ ppm; 0.7-1.0 (6H, m, CH(CH₃)₂ ), 1.1-2.1 (12H, m, (CH₃)₂CH—CH₂ ,+C(CH₃)₃ ), 2.8 (2H, bd, J=5.4 Hz, CH₂ —CH═CH), 3.0-3.3 (1H, m,CH₂—CH—CO), 5.0-5.2 (4H, m, CH₂ —O×2), 6.1-6.4 (2H, m, CH₂—CH═CH),7.1-7.5 (15H, m, aromatic-H).

(d) 3(RS)-tert-butoxycarbonyl-6-phenyl-2(R)-isobutylhexanoic Acid

The compound (4.2 g, 7.36 mmol) of Example 3-c was dissolved in ethanol(35 ml), added with 10% Pd-C (50% wet, 1.3 g), and then stirredvigorously in a stream of hydrogen at room temperature for 7 hours.After the catalyst was filtered off, ethanol of the filtrate was removedunder reduced pressure. The residue was added with N-methylmorpholine(0.81 ml, 7.36 mmol) and toluene (50 ml), and refluxed for 2 hours. Thereaction liquid was washed with 1N-HCl and then a saturated aqueoussolution of NaCl two times respectively, dried over anhydrous MgSO₄,evaporated under reduced pressure and purified by column chromatography(silica gel; 150 g, elution with the mixed solvent; CHCl₃:MeOH=200:1),giving the title compound (1.1 g, 43%) of a colorless oil.

Rf value; 0.60 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(CDCl₃) δ ppm; 0.88 (6H, bd, J=5.7 Hz, CH(CH₃)₂ ), 1.0-2.0 (16H,m, (CH₃)₂ CH—CH₂, +C(CH)₃)₃ +CH₂—CH₂ —CH₂—Ph), 2.4-2.8 (4H, m,CH—CO×2+CH₂ —Ph), 7.0-7.4 (5H, m, aromatic-H).

(e) N-[4-tert-Butoxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-tyrosine-N-methylamide

The compound (4.0 g, 11.5 mmol) of Example 3-d, L-tyrosine-N-methylamidehydrochloride (4.0 g, 17.3 mmol) and HOBT (1.60 g, 11.8 mmol) weredissolved in DMF (25 ml) and CH₂Cl₂ (25 ml) and added with TEA (2.50 ml,17.9 mmol) and EDC (2.65 g, 13.8 mmol) at −15° C. under stirring. Themixture was stirred at −15° C. for 1 hour and further at roomtemperature overnight and then evaporated under reduced pressure. Theresidue was added with AcOEt (100 ml), washed with a saturated aqueoussolution of NaCl, 1N-HCl, a saturated aqueous solution of NaHCO₃ andthen the saturated aqueous solution of NaCl two times respectively,dried over anhydrous MgSO₄, evaporated under reduced pressure andpurified by column chromatography (silica gel; 150 g, elution with themixed solvent; n-hexane:AcOEt=10:9), giving two isomers of the titlecompounds as pale yellow oils.

Isomer A

The pale yellow oil (30%), Specific rotation[α]_(D)=−8.2° (c=0.5, MeOH),Rf value; 0.30 (AcOEt:n-hexane=1:1).

¹H-NMR(CDCl₃) δ ppm; 0.80 (6H, m, CH(CH₃)₂ ), 0.9-1.9 (16H, s+m, (CH₃)₂CH—CH₂, +C(CH₃)₃ +CH₂—CH₂ —CH₂—Ph), 2.2-3.1 (9H, m, N—CH₃ +—CH—CO×2+CH₂—Ph+C₆H₄ CH₂ ), 4.54 (1H, m, NH—CH—CO), 6.0 (1H, m, NH), 6.4-7.4 (9H, m,aromatic-H).

Isomer B

The pale yellow oil (38%), specific rotation[α]_(D)=+9.0° (c=0.5, MeOH),Rf value; 0.29 (AcOEt:n-hexane=1:1).

¹H-NMR(CDCl₃) δ ppm; 0.75 (6H, d, J=4.6 Hz, CH(CH₃)₂ ) 0.8-1.7 (16H,s+m, (CH₃)₂ CH—CH_(2,) +C(CH₃)₃ +CH₂—CH₂ —CH₂—Ph), 2.1-3.1 (9H, d+m,J=4.6 Hz, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ ), 4.65 (1H, m, NH—CH—CO),6.3-7.4 (11H, m, NH×2+aromatic-H).

The following compounds were synthesized in the same methods as used ind-g of Example 1.

(f) N-[4-Hydroxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-tyrosine-N-methylamide

Isomer B

White solid (89%), m.p.; 222° C., specific rotation[α]_(D)=−7.2° (c=0.5,MeOH), Rf value; 0.38 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR (MeOH-d₄) δ ppm; 0.78 (6H, m, CH(CH₃)₂ ), 0.9-1.6 (7H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 2.1-3.0 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄CH₂ ), 4.60 (1H, m, NH—CH—CO), 6.3-7.4 (9H, m, aromatic-H).

Isomer A

White solid (85%), m.p.; 173-177° C., specific rotation[α]_(D)=+8.1°(c=1.0, MeOH), Rf value; 0.39 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR (MeOH-d₄) δ ppm; 0.85 (6H, m, CH(CH₃)₂ ), 0.9-1.8 (7H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 2.2-3.2 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄CH₂ ), 4.50 (1H, m, NH—CH—CO), 6.4-7.5 (9H, m, aromatic-H).

(g) N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-tyrosine-N-methylamide

Isomer B (yield 60%)

m.p.; 220° C., specific rotation[α]_(D)=+5.60° (c=1.0, DMF), Rf value;0.51 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(MeOH-d₄) δ ppm; 0.8 (6H, m, CH(CH₃)₂ ), 0.9-1.6 (7H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 2.0-3.0 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄CH₂ ), 4.60 (1H, m, NH—CH—CO), 6.3-7.5 (14H, m, aromatic-H).

Isomer A (yield 50%)

m.p.; 185-190° C., specific rotation[α]_(D)=+8.2° (c=0.5, MeOH), Rfvalue; 0.51 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(MeOH-d₄) δ ppm; 0.8-1.8 (13H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph),2.2-3.2 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ ), 4.5 (1H, m,NH—CH—CO), 6.5-7.5 (14H, m, aromatic-H).

(h) N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-methylamide,Monosodium Salt

Isomer B

White amorphous powder (92%), specific rotation[α]_(D)=+10.0° (c=0.9,MeOH), Rf value; 0.86 (CHCl₃:MeOH:AcOH=5:2:1), 0.82(n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.7 (13H, m, J=4.6 Hz, (CH₃)₂CH—CH₂ +CH₂—CH₂—CH₂—Ph), 1.9-3.0 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ ), 4.50 (1H,m, NH—CH—CO), 7.1-7.5 (14H, m, aromatic-H).

Isomer A

White amorphous powder (95%), specific rotation[α]_(D)=+13.5° (c=1.0,MeOH), Rf value; 0.86 (CHCl₃:MeOH:AcOH=5:2:1), 0.82(n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.6-1.8 (13H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph),2.0-3.0 (9H, m, N—CH₃ +—CH—CO×2+CH—Ph+C₆H₄ CH₂ ), 4.47 (1H, m,NH—CH—CO), 7.0-7.5 (14H, m, aromatic-H).

(i) N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-methylamide,Monosodium Salt

Isomer B

White amorphous powder (98%), specific rotation[α]_(D)=−41.0° (c=1.0,MeOH), Rf value; 0.69 (CHCl₃:MeOH:AcOH=5:2:1), 0.76(n-BuOH:AcOH:water=4:1:1). Analytical value calculated forC₂₇H₃₆N₃O₈SNa. Theoretical value:C, 55.37; H, 6.20; N, 7.17. Found:C,55.37; H, 6.39; N, 7.24.

¹H-NMR(MeOH-d₄) δ ppm; 0.6-1.7 (13H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—PH),1.8-3.1 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ ), 4.50 (1H, m,NH—CH—CO), 7.22 (9H, m, aromatic-H).

Isomer A

White amorphous powder (100%), specific rotation[α]_(D)=−11.9° (c=1.0,MeOH), Rf value; 0.69 (CHCl₃:MeOH:AcOH=5:2:1), 0.77(n-BuOH:AcOH:water=4:1:1). Analytical value calculated forC₂₇H₃₆N₃O₈SNa. Theoretical value:C, 55.37; H, 6.20; N, 7.17. Found:C,55.45; H, 6.29; N, 7.36.

¹H-NMR(MeOH-d₄) δ ppm; 0.6-1.8 (13H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph),2.0-3.4 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ ), 4.60 (1H, m,NH—CH—CO), 7.2 (9H, m, aromatic-H).

EXAMPLE 4N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

(a)N-[4-tert-Butoxy-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

By the same method as that of Example 2-c, the title compound (3.69 g,60%) of a white solid was obtained from the compound (3.49 g, 10.0 mmol)of Example 3-d and the compound (3.64 g, 13.0 mmol) of Example 2-b.

m.p.; 235-240° C., specific rotation[α]_(D)=−6.97° (c=1.0, MeOH), Rfvalue; 0.66 (CHCl₃:MeOH:AcOH=5:2:1), 0.71 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.55-1.06 (6H, m, CH—(CH₃)₂ ), 1.1-1.8 (16H, m,C(CH₃)₃ +CH₂CH(CH₃)₂+PhCH₂ (CH₂)₂ ), 2.2-2.8 (7H, m, PhCH₂ +NHCH₃+CO—CH×2), 2.81-3.2 (2H, m, C₆H₄—CH₂ ), 4.58 (1H, m, NH—CH—CO), 6.8-7.9(9H, m, aromatic-H).

(b)N-[4-Hydroxy-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

To the compound (3.84 g, 6.29 mmol) of Example 4-a was added ice-cooled95% aqueous trifluoroacetic acid (45 ml). The reaction mixture wasstirred at 50° C. overnight and concentrated under reduced pressure. Theresidue was added with Et₂O and stirred at room temperature for 1 hour.The precipitating solid was filtered and dried in a desiccator underreduced pressure.

m.p.; 243-256° C., specific rotation[α]_(D)=−4.95° (c=1.0, MeOH), Rfvalue; 0.53 (CHCl₃:MeOH:AcOH=5:2:1), 0.67 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.0 (6H, m, CH—(CH₃)₂ ), 1.2-1.8 (7H, m,CH₂CH(CH₃)₂+PhCH₂ (CH₂)₂ ), 2.2-2.7 (7H, m, PhCH₂ +NHCH₃ +CO—CH×2),2.8-3.2 (2H, m, C₆H₄—CH₂ ) 4.56 (1H, bm, NH—CH—CO), 6.8-7.9 (9H, m,aromatic-H).

(c)N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (3.53 g, 6.37 mmol) of Example 4-b and HOBT (1.29 g, 9.56mmol) were dissolved in DMF (180 ml), added with EDC (1.83 g, 9.56 mmol)at an ice-cooling temperature under stirring and stirred at anice-cooling temperature for 1 hour. The mixture was added withhydroxylamine hydrochloride (664 mg, 9.56 mmol) and dropped withtriethylamine (1.33 ml, 9.56 mmol), stirred under ice-cooling for 3hours and evaporated under reduced pressure. The residue was purified bya reverse-phase column chromatography (Fuji Silysia Chemical Ltd.,Chromatorex ODS DM-1020T; 250 g, elution with a MeOH-water solutioncontaining 5-20% MeOH) to give the title compound (1.57 g, 43%) of whiteamorphous powder upon freeze-drying.

Specific rotation[α]_(D)=−3.73° (c=1.0, MeOH), Rf value; 0.42(CHCl₃:MeOH:AcOH=5:2:1), 0.66 (n-BuOH:AcOH:water 4:1:1), Analyticalvalue calculated for C₂₇H₃₆N₃O₇SNa. Theoretical value:C, 56.93; H, 6.37;N, 7.38. Found:C, 56.69; H, 6.58; N, 7.1.

¹H-NMR(MeOH-d₄) δ ppm; 0.8-0.95 (6H, m, CH—(CH₃)₂ ), 0.96-1.82 (7H, m,CH₂CH(CH₃)₂+PhCH₂ (CH₂)₂ ), 1.82-2.74 (7H, m, PhCH₂ +NHCH₃ +2×CO—CH),2.75-3.5 (2H, m, C₆H₄—CH₂ ), 4.56 (1H, brm, NH—CH—CO), 6.95-7.40 (9H, m,aromatic-H).

EXAMPLE 5 N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(thienylthiomethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

(a)N-[4-Benzyloxy-3-benzyloxycarbonyl-2(R)-isobutyl-succinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

By the same method as that of Example 2-c, the title compound (12.6 g,65%) of white crystals was obtained from the compound (8.18 g, 29.2mmol) of Example 2-b and2(R)-isobutyl-3,3-dibenzyloxycarbonyl-n-propionic acid (11.6 g, 29.2mmol).

m.p.; 237-245° C., specific rotation[α]_(D)=−14.0° (c=1.0, MeOH), Rfvalue; 0.79 (CHCl₃:MeOH:AcOH=5:2:1), 0.60 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.6-0.9 (6H, m, CH(CH₃)₂ ), 0.95-1.7 (3H, m,CH₂CH(CH₃)₂), 2.5-3.8 (m, C₆H₄ CH₂ +NHCH₃ +CO—CHCH—CO), 4.3-4.8 (1H, m,NH—CH—CO), 5.0-5.2 (4H, m, PhCH₂ ×2), 7.2-7.9 (14H, m, aromatic-H).

(b)N-[4-Hydroxy-3-hydroxycarbonyl-2(R)-isobutylsuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (12.6 g, 19.1 mmol) of Example 5-a was dissolved in MeOH(250 ml), added with 10% Pd-C (50% wet, 12.6 g), and then stirredvigorously in a stream of hydrogen at room temperature for 5 hours.After the catalyst was filtered off, MeOH of the filtrate was removedunder reduced pressure. The concentrate was lyophilized, whereby thetitle compound (8.92 g, 97%) of white amorphous powder was obtained.

Specific rotation[α]_(D)=+21.0° (c=1.0, MeOH), Rf value; 0.16(CHCl₃:MeOH:AcOH=5:2:1), 0.29 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.75-0.95 (6H, m, CH(CH₃)₂ ), 0.95-1.7 (3H, m,CH₂CH(CH₃)₂), 2.8-3.2 (6H, m, C₆H₄ CH₂ +NHCH₃ +(HO₂C)₂CH—CH), 3.55 (1H,d, (HO₂C)₂ CH), 4.53 (1H, m, NH—CH—CO), 4.97 (2H, brm, 2×NH), 7.2-8.9(4H, AA′BB′, aromatic-H).

(c)N-[4-Hydroxy-2(R)-isobutyl-3-ethenylsuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (8.72 g, 18.2 mmol) of Example 5-b and piperidine (3.40 g,39.9 mmol) were dissolved in ethanol (90 ml), stirred at roomtemperature for 30 min. and added with 36% formalin solution (14.9 ml,181 mmol). The reaction mixture was stirred at room temperature for 1hour and further at 80° C. for 1 hour and concentrated under reducedpressure. The residue was dissolved in 1N-HCl (200 ml) and extracted twotimes with THF (200 ml). The THF solution was washed with a saturatedaqueous solution of NaCl, dried over anhydrous MgSO₄ and evaporatedunder reduced pressure to give the title compound (7.90 g, 97%) of apale yellow solid.

¹H-NMR(MeOH-d₄+CDCl₃) δ ppm; 0.75-1.0 (6H, m, CH(CH₃)₂ ), 1.1-2.0 (3H,m, CH₂CH(CH₃)₂), 2.70 (3H, s, NHCH₃ ), 2.9-3.2 (2H, m, C₆H₄ CH₂ ),3.4-3.8 (1H, m, HO₂C—C—CH), 4.45-4.7 (1H, m, NH—CH—CO), 5.12 (2H, brm,2×NH), 5.63, 6.25 (2×1H, 2×s, C═CH₂ ), 7.15-7.85 (4H, AA′BB′,aromatic-H).

(d)N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3-ethenylsuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (7.90 g, 17.6 mmol) of Example 5-c, O-benzylhydroxylaminehydrochloride (11.3 g, 70.5 mmol) and HOBT (5.24 g, 38.8 mmol) weresuspended in DMF (320 ml). The suspension was added with TEA (7.13 ml,70.5 mmol) and EDC (10.1 g, 52.8 mmol) at −15° C. under stirring andstirred at room temperature overnight. The reaction mixture wasconcentrated under reduced pressure and purified by a reverse-phasecolumn chromatography (Fuji Silysia Chemical Ltd., ChromatorexODS-1020T; 200 g, elution with 30-36% MeOH water) to give the titlecompound (4.10 g, 42%) of white amorphous powder upon freeze-drying.

m.p.; 102-106° C., specific rotation[α]_(D)=−13.8° (c=1.0, MeOH), Rfvalue; 0.71 (CHCl₃:MeOH:AcOH=5:2:1), 0.60 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.75-0.95 (6H, m, CH(CH₃)₂ ), 1.0-2.0 (3H, m,CH₂CH(CH₃)₂), 2.6-3.2 (6H, m, NHCH₃ +C₆H₄ CH₂ +CO—C—CH), 4.5-4.75 (1H,m, NH—CH—CO), 4.85 (3H, brm, 3×NH), 4.90 (2H, s, PhCH₂ ), 5.23, 5.48(2×1H, 2×s, C═CH₂ ), 7.1-7.8 (4H, AA′BB′, aromatic-H).

(e)N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(RS)-(thienylthiomethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (4.10 g, 7.41 mmol) of Example 5-d was dissolved inthiophenethiol (23.7 g, 204 mmol), and stirred in a dark and nitrogenatmosphere at 60° C. overnight. The reaction mixture was added withEt₂O. The resulting crystals were filtered and washed with Et₂O to givethe title compound (4.23 g, 86%) of yellow crystals upon drying.

m.p.; 221-226° C. (dec.), specific rotation[α]_(D)=−35.0° (c=1.0, MeOH),Rf value; 0.71 (CHCl₃:MeOH:AcOH=5:2:1), 0.69 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄+CDCl₃) δ ppm; 0.5-1.7 (9H, m, (CH₃)₂CH—CH₂ ), 1.8-3.6(9H, m, N—CH₃ +S—CH₂CHCH—CO+C₆H₄ CH₂ ), 4.4-4.7 (3H, m, PhCH₂ +N—CH—CO),6.85-7.9 (12H, m, aromatic-H).

(f) N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(thienylthiomethylene)succinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

The compound (4.23 g, 6.34 mmol) of Example 5-e was added with anisole(5 ml) and hydrofluoric acid (50 ml) and stirred at 0° C. for 30 min.The hydrofluoric acid was evaporated under reduced pressure. The residuewas added with Et₂O. The resulting crystals were purified by areverse-phase column chromatography (Fuji Silysia Chemical Ltd.,Chromatorex ODS-1020T; 200 g, elution with a MeOH-water solutioncontaining 9-13% MeOH) to give the title compound (1.33 g, 36.4%) of twokinds as white amorphous powder upon freeze-drying.

Isomer A (1.33 g, 36%)

m.p.; 142-148° C., specific rotation[α]_(D)=−44.9° (c=1.0, MeOH), Rfvalue; 0.44 (CHCl₃:MeOH:AcOH=5:2:1), 0.51 (n-BuOH:AcOH:water=4:1:1).Analytical value calculated for C₂₃H₃₀N₃O₇S₃Na. Theoretical value:C,47.66; H, 5.22; N, 7.25. Found:C, 47.4; H, 5.44; N, 7.03.

¹H-NMR(MeOH-d₄) δ ppm; 0.7-0.95 (6H, m, (CH₃)₂ CH—CH₂), 1.0-1.7 (3H, m,CH₂—CH(CH₃)₂), 1.8-3.6 (9H, m, N—CH₃ +S—CH₂CHCH—CO+C₆H₄ CH₂ ), 4.4-4.7(1H, m, NH—CH—CO), 4.85 (3H, brm, 3×NH), 6.9-7.9 (7H, m, aromatic-H).

Isomer B (0.40 g, 11%)

m.p.; 132-137° C., specific rotation[α]_(D)=+43.2° (c=1.0, MeOH), Rfvalue; 0.57 (CHCl₃:MeOH:AcOH=5:2:1), 0.56 (n-BuOH:AcOH:water=4:1:1).Analytical value calculated for C₂₃H₃₀N₃O₇S₃Na. Theoretical value:C,47.66; H, 5.22; N, 7.25. Found:C, 47.39; H, 5.5; N, 7.11.

¹H-NMR(MeOH-d₄) δ ppm; 0.7-0.95 (6H, m, (CH₃)₂ CH—CH₂), 1.0-1.7 (3H, m,CH₂—CH(CH₃)₂), 1.8-3.6 (9H, m, N—CH₃ +S—CH₂CHCH—CO+C₆H₄ CH₂ ), 4.4-4.7(1H, m, NH—CH—CO), 4.85 (3H, brm, 3×NH), 6.9-7.9 (7H, m, aromatic-H).

EXAMPLE 6N-[4-(N-Hydroxyamino)-2(R)-n-propyloxymethylene-3(RS)-isopropylthiomethylenesuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

(a) Benzyl 2(R)-bromo-3-n-propyloxypropionic Acid

By the same method as that of Example 3-a, the title compound (1.11 g,67%) of a colorless oil was obtained from2(R)-bromo-3-n-propyloxypropionic acid (1.16 g, 5.52 mmol).

Specific rotation[α]_(D)=+0.89° (c=1.2, MeOH), Rf value; 0.61(n-hexane:AcOEt=5:1).

¹H-NMR(CDCl₃) δ ppm; 0.88 (3H, t, J=7.7 Hz, CH₃ ), 1.3-1.8 (2H, m, CH₃CH₂ ), 3.42 (2H, t, J=6.2 Hz, O—CH₂ ), 3.82 (2H, m, CH₂ —O), 4.35 (1H,m, CH), 5.22 (2H, s, CH₂ Ph), 7.38 (5H, s, aromatic-H).

(b) Dibenzyl3(RS)-tert-butoxycarbonyl-2(R)-n-propyloxymethylenesuccinate

By the same method as that of Example 3-b, the title compound (22.9 g,71%) of colorless crystals was obtained from the compound (20.4 g, 67.9mmol) of Example 6-a.

m.p.; 31-33° C., specific rotation[α]_(D)=+3.22° (c=1.1, MeOH), Rfvalue; 0.45 (n-hexane:AcOEt=5:1).

¹H-NMR(CDCl₃) δ ppm; 0.82 (3H, 2×t, J=7.7 Hz, CH₃ ), 1.1-1.7 (11H,2×s+m, CH₃ CH₂ +(CH₃)₃ C), 3.1-3.8 (5H, m, CH₂ —O—CH₂ , CH₂—CH—CO), 3.91(1H, d, CO—CH—CO), 5.05-5.22 (4H, m, 2×CH₂ Ph), 7.35 (10H, s,aromatic-H).

(c) 3-tert-Butoxycarbonyl-2(R)-n-propyloxymethylene-3-butenoic Acid

The compound (3.00 g, 6.38 mmol) of Example 6-b was dissolved in MeOH(100 ml), added with 10% Pd-C (50% wet, 1.5 g) and then stirredvigorously in a stream of hydrogen at room temperature for 6 hours.After the catalyst was filtered off, the filtrate was added withpiperidine (694 μl, 7.02 mmol) and stirred at room temperature 15 min.The reaction liquid was added with 36% formalin solution (3.86 ml, 47.5mmol) at room temperature and stirred at room temperature overnight andfurther at 80° C. 1 hour. After the solvent was removed under reducedpressure, the residue was dissolved in AcOEt and washed with 5% aqueouscitric acid and then a saturated aqueous solution of NaCl two timesrespectively. The organic layer was dried over anhydrous MgSO₄,evaporated under reduced pressure to remove the solvent and purified bycolumn chromatography (silica gel; 50 g, elution with the mixed solvent;CH₂Cl₂:AcOEt=7:1) to give the title compound (1.09 g, 66%) of acolorless oil.

¹H-NMR(CDCl₃) δ ppm; 0.89 (3H, t, J=7.7 Hz, CH₂ CH₃ ), 1.1-1.75 (11H,s+m, CH₃ CH₂ +(CH₃)₃ C), 3.41 (2H, t, J=6.3 Hz, O—CH₂ —CH₂), 3.57-3.98(3H, m, CO—CHCH₂ —O), 5.77, 6.34 (2×1H, 2×s, C═CH₂ ), 9.2 (1H, brs, OH).

(d)4-Acetylthio-3(RS)-tert-butoxycarbonyl-2(R)-n-propyloxymethylenebutanoicAcid

The compound (700 mg, 2.71 mmol) of Example 6-c and thioacetic acid (5ml, 70.6 mmol) were mixed and stirred at room temperature overnight. Thereaction mixture was purified by column chromatography (silica gel; 100g, elution with the mixed solvent; CH₂Cl₂:MeOH=50:1) to give the titlecompound (823 mg, 91%) of a colorless oil.

Specific rotation[α]_(D)=−1.74° (c=0.60, MeOH), Rf value; 0.56(CHCl₃:MeOH=10:1).

¹H-NMR(CDCl₃) δ ppm; 0.90 (3H, t, J=7.8 Hz, CH₂ CH₃ ), 1.1-1.75 (11H,s+m, CH₃ CH₂ +(CH₃)₃ C), 2.22 (3H, s, CH₃ CO), 2.80-3.55 (6H, m, SCH₂+CH₂ —O—CH₂ ), 3.55-3.90 (2 H, m, 2×CO—CH).

(e)3(RS)-tert-Butoxycarbonyl-4-isopropylthio-2(R)-n-propyloxymethylenebutanoicAcid

The compound (823 mg, 2.46 mmol) of Example 6-d was dissolved in MeOH(14 ml) under nitrogen atmosphere and dropped with 1N-NaOH (12.3 ml) atroom temperature under stirring. After 25 min., the mixture was addedwith isopropyl iodide (982 μl, 9.84 mmol) and stirred vigorously at roomtemperature. The reaction mixture was neutralized with 1N-HCl,evaporated under reduced pressure to remove MeOH and acidified by 1N-HClto pH=2. The organic substances were extracted with AcOEt and washedwith a saturated aqueous solution of NaCl two times. The organic layerwas dried over anhydrous MgSO₄, evaporated under reduced pressure andpurified by column chromatography (silica gel; 50 g, elution with themixed solvent; CHCl₃:MeOH=60:1) to give the title compound (653 mg, 80%)of a colorless oil.

Specific rotation[α]_(D)=+0.94° (c=1.1, MeOH), Rf value; 0.46(CHCl₃:MeOH=10:1).

¹H-NMR(CDCl₃) δ ppm; 0.89 (3H, t, J=7.7 Hz, CH₂ CH₃ ), 1.24 (6H, 2×d,J=7.7 Hz, CH—(CH₃)₂ ), 1.1-1.80 (11H, s+m, CH₃ CH₂ +(CH₃)₃ C), 2.60-3.27(5H, m, O—CH₂ —CH+CH—S—CH₂ ), 3.40 (2H, t, J=6.3 Hz, O—CH₂ —CH₂),3.55-3.80 (2 H, m, 2×CO—CH).

(f)N-[4-tert-Butoxy-3(RS)-isopropylthiomethylene-2(R)-n-propyloxymethylenesuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

By the same method as that of Example 2-c, the title compound (653 mg,80%) of colorless crystals was obtained from the compound of Example6-e.

m.p.; 254° C. (dec.), specific rotation[α]_(D)=+3.26° (c=0.70, MeOH), Rfvalue; 0.64 (CHCl₃:MeOH:AcOH=5:2:1), 0.65 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR (MeOH-d₄) δ ppm; 0.70-1.02 (3H, m, CH₂ CH₃ ), 1.24 (6H, 2×d,J=7.7 Hz, CH—(CH₃)₂ ), 1.1-1.8 (11H, s+m, CH₃ CH₂ +(CH₃)₃ C), 2.5-3.8(14H, m, NHCH₃ +C₆H₄—CH₂ +CH₂ —O—CH₂ +CH—S—CH₂ +2×CO—CH), 4.5 (1H, m,NH—CH—CO), 7.53 (4H, AA′BB′, aromatic-H).

(g)N-[4-Hydroxy-3(RS)-isopropylthiomethylene-2(R)-n-propyloxymethylenesuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

To the compound (1.32 g, 2.22 mmol) of Example 6-f was added ice-cooled95% aqueous trifluoroacetic acid (40 ml). The reaction mixture wasstirred at 5° C.for 2 hours and concentrated under reduced pressure. Theresidue was added with Et₂O and stirred at room temperature for 1 hour.The precipitating solid was purified by a reverse-phase columnchromatography (Fuji Silysia Chemical Ltd., Chromatorex ODS DM-1020T; 90g, elution with a MeOH-water solution containing 5-15% MeOH) to give thetitle compound (389 mg, 32%) of colorless powder.

m.p.; 127-137° C., specific rotation[α]_(D)=−8.49° (c=0.99, MeOH), Rfvalue; 0.48 (CHCl₃:MeOH:AcOH=5:2:1), 0.59 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.9 (3H, m, CH₂ CH₃ ), 1.22 (6H, 2×d, J=7.8 Hz,CH—(CH)₃)₂ ), 1.4-1.75 (2H, m, CH₃ CH₂ ), 2.6-3.8 (14H, m, NHCH₃+C₆H₄—CH₂ +CH₂ —O—CH₂ +CH—S—CH₂ +2×CO—CH), 4.55 (1H, m, NH—CH—CO), 7.53(4H, AA′BB′, aromatic-H).

(h)N-[4-(N-Hydroxyamino)-3(RS)-isopropylthiomethylene-2(R)-n-propyloxymethylenesuccinyl]-L-4′-sulfophenylalanine-N-methylamide,Monosodium Salt

By the same method as that in Example 4-c, the title compound (236 mg,62%) of a white solid was obtained from the compound (370 mg, 0.686mmol) of Example 6-g.

m.p.; l01-107° C., specific rotation[α]_(D)=−2.18° (c=0.53, MeOH), Rfvalue; 0.41 (CHCl₃:MeOH:AcOH=5:2:1), 0.56 (n-BuOH:AcOH:water=4:1:1).Analytical value calculated for C₂₂H₃₄N₃O₇S₂Na. Theoretical value:C,48.97; H, 6.35; N, 7.79. Found:C, 48.79; H, 6.51; N, 7.62.

¹H-NMR(MeOH-d₄) δ ppm; 0.9 (3H, m, CH₂ CH₃ ), 1.22 (6H, 2×d, J=7.7 Hz,CH—(CH₃)₂ ), 1.4-1.75 (2H, m, CH₃ CH₂ ), 2.6-3.8 (14H, m, NHCH₃+C₆H₄—CH_(2—) +CH₂ —O—CH₂ +CH—S—CH₂ +2×CO—CH), 4.55 (1H, m, NH—CH—CO),7.53 (4H, AA′BB′, aromatic-H).

EXAMPLE 7N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-p-methoxyphenylamide,Monosodium Salt

By the same methods as used in c-g of Example 1, the following compoundswere synthsized from the compound of Example 3-d andL-tyrosine-N-p-methoxyphenylamide hydrochlodide.

(a)N-[4-tert-Butoxy-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-tyrosine-N-p-methoxyphenylamide

White solid (86%), m.p.; 87-89° C., specific rotation[α]_(D)=−27.0°(c=1.02, CHCl₃), Rf value; 0.56 (AcOEt:n-hexane=1:1).

¹H-NMR(CDCl₃) δ ppm; 0.77 (6H, m, CH(CH₃)₂ ), 0.9-1.8 (16H, s+m, (CH₃)₂CH—CH₂ +C(CH₃)₃ +CH₂—CH₂ —CH₂—Ph), 2.1-2.7 (4H, m, —CH—CO×2+CH₂ —Ph),2.8-3.2 (2H, m, C₆H₄ CH₂ ), 3.74 (3H, s, OCH₃ ), 4.78 (1H, m, NH—CH—CO),6.3-7.6 (13H, m, aromatic-H).

(b)N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-tyrosine-N-p-methoxyphenylamide

White powder (41%), m.p.; 241-244° C., specific rotation[α]_(D)=+9.39°(c=1.0, DMF), Rf value; 0.24 (CHCl₃:MeOH=20:1).

¹H-NMR(DMSO-d₆) δ ppm; 0.4-1.6(13H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph),1.8-2.6 (4H, m, —CH—CO×2+CH₂ —Ph), 2.92 (2H, m, C₆H₄ CH₂ ), 3.72 (3H, s,OCH₃ ), 4.4-4.8 (3H, s+m, O—CH₂ —Ph+NH—CH—CO), 6.5-7.6 (18H, m,aromatic-H), 8.32 (1H, m, NH).

(c)N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-p-methoxyphenylamide

White amorphous powder (71%), specific rotation[α]_(D)=−16.8° (c=1.0,MeOH), Rf value; 0.66 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(DMSO-d₆) δ ppm; 0.5-1.0 (6H, m, (CH₃ ₂ CH—CH₂), 1.0-1.7 (7H, m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.8-2.7 (4H, m, —CH—CO×2+CH₂ —Ph),2.8-3.2 (2H, m, C₆H₄ CH₂ ), 3.74 (3H, s, OCH₃ ), 4.5-5.1 (3H, s+m, O—CH₂—Ph+NH—CH—CO), 6.2-7.6 (18H, m, aromatic-H).

(d)N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-p-methoxyphonylamide,Monosodium Salt

White amorphous powder (73%), specific rotation[α]_(D)=−7.4° (c=1.1,DMF), Rf value; 0.45 (CHCl₃:MeOH:AcOH=5:2:1). Analytical valuecalculated for C₃₃H₄₀N₃O₉SNa. Theoretical value:C, 58.48; H, 5.95; N,6.2. Found:C, 58.27; H, 6.2; N, 6.01.

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.8 (13H, m, (CH₃)CH₂CH—CH₂ +CH₂—CH₂—CH₂—Ph), 1.9-2.7 (4H, m, —CH—CO×2+CH₂ —Ph), 2.8-3.2 (2H, m, C₆H₄ CH₂ ),3.73 and 3.75 (3H, s each, OCH₃ ), 4.5-5.0 (1H, m, NH—CH—CO), 6.6-7.6(18H, m, aromatic-H).

EXAMPLE 8 N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-guanidophenylalanine-N-methylamideMonoacetate

(a)N^(α)-tert-Butyloxycarbonyl-L-4′-[N,N′-bis(benzyloxycarbonyl)guanido]phenylalanine-N-methylamide

N^(α)-tert-Butyloxycarbonyl-L-4′-aminophenylalanine-N-methylamide (2.00g, 6.82 mmol) was dissolved in CH₂Cl₂ (30 ml), added with1H-pyrazole-N,N′-bis (benzyloxycarbonyl) carboxamidine (2.84 g, 7.51mmol) and stirred at room temperature for 3 hours. The reaction mixturewas concentrated under reduced pressure, dissolved in AcOEt (30 ml) andstirred for 30 min. The resulting crystals were dried to give the titlecompound (3.95 g, 96%) of a white solid.

m.p.; 168° C., specific rotation[α]_(D)=+14.1° (c=1.0, CHCl₃), Rf value;0.20 (CHCl₃:MeOH=50:1).

¹H-NMR(CDCl₃) δ ppm; 1.40 (9H, s, C(CH₃)₃ ), 2.69 (3H, d, J=4.8 Hz,N—CH₃ ), 3.00 (2H, d, J=6.8 Hz, C₆H₄ CH₂ ), 4.9-5.4 (5H, m+s, OCH₂Ph×2+NH), 6.00 (1H, m, NH), 7.0-7.7 (16H, m, aromatic-H+NH×2).

(b) N-[4-tert-Butoxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-[N,N′-bis(benzyloxycarbonyl)guanido]phenylalanine-N-methylamide

The compound (142 g, 235 mmol) of Example 8-a was dissolved in an AcOEtsolution of HCl (4N, 1000 ml) and stirred under ice-cooling for 45 min.The reaction mixture was added with Et₂O (150 ml) and stirred. Theprecipitating crystals were filtered, washed with Et₂O (100 ml) fivetimes, and dried.

The obtained crystals (127 g, 235 mmol), the compound (73.9 g, 212 mmol)of Example 3-d and HOBT (30.1 g, 223 mmol) were dissolved in DMF (50 ml)and CH₂Cl₂ (1200 ml) and added with TEA (32.8 ml, 235 mmol) and EDC(44.7 g, 233 mmol) at −15° C. under stirring. The mixture was stirred at−15° C. for 1 hour and further at room temperature overnight andevaporated under reduced pressure. The residue was purified by columnchromatography (silica gel; 12 Kg, elution with the mixed solvent;CH₂Cl₂:AcOEt=6:1-2:1) to give the title compound (isomer A; 65.0 g, 37%,and isomer B; 58.0 g, 33%) of a white solid.

Isomer A

m.p.; 191° C., specific rotation[α]_(D)=−10.1° (c=1.0, CHCl₃), Rf value;0.28 (n-hexane:AcOEt=1:1).

¹H-NMR(CDCl₃) δ ppm; 0.82 (6H, m, CH(CH(₃)₂ ), 0.92-2.1 (16H, s+m,(CH₃)₂ CH—CH₂, +C(CH₃)₃ +CH₂—CH₂ —CH₂—Ph), 2.1-2.6 (4H, m, —CH—CO×2+CH₂—Ph), 2.67 (3H, d, J=4.6 Hz, N—CH₃ ), 2.93 (2H, d, J=5.9 Hz, C₆H₄ CH₂ ),4.56 (1H, m, NH—CH—CO), 5.13 and 5.18 (4H, s each, OCH₂ Ph×2), 5.98 (1H,m, NH), 6.50 (1H, m, NH), 6.8-7.65 (21H, m, aromatic-H+NH×2).

Isomer B

m.p.; 160° C., specific rotation[α]_(D)=+10.70° (c=1.0, CHCl₃), Rfvalue; 0.37 (n-hexane:AcOEt=1:1).

¹H-NMR(CDCl₃) δ ppm; 0.80 (6H, m, CH(CH₃)₂ ), 0.9-1.95 (16H, s+m, (CH₃)₂CH—CH₂, +C(CH₃)₃ +CH₂—CH₂ —CH₂—Ph), 2.0-2.6 (4H, m, —CH—CO×2+CH₂ —Ph),2.68 (3H, d, J=4.6 Hz, N—CH₃ ), 3.01 (2H, d, J=5.9 Hz, C₆H₄ CH₂ ), 4.61(1H, m, NH—CH—CO), 5.15 and 5.23 (4H, s each, OCH₂ Ph×2), 5.8-6.3 (2H,m, NH×2), 6.95-7.75 (21H, m, aromatic-H+NH×2).

(c) N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-[N,N′-bis(benzyloxycarbonyl)guanido]phenylalanine-N-methylamide

To the compound (isomer A, 56.0 g, 67.1 mmol) of Example 8-b was addedice-cooled 95% aqueous trifluoroacetic acid (420 ml). The reactionmixture was stirred at 5° C. overnight and concentrated under reducedpressure. The residue was added with Et₂O and stirred at roomtemperature for 1 hour. The precipitating solid was filtered and driedto give a white solid (54.7 g).

The obtained white solid, O-benzylhydroxylamine hydrochloride (16.8 g,105 mmol) and HOBT (10.5 g, 77.3 mmol) were suspended in DMF (1400 ml).The suspension was added with TEA (15.2 ml, 109 mmol) and EDC (20.1 g,105 mmol) at −15° C. under stirring. The mixture was stirred at −15° C.for 1 hour and further at room temperature overnight and dropped withice-water (3000 ml). The precipitating solid was filtered, suspended inMeOH (3000 ml) and stirred at room temperature for 1 hour. The insolublematerial was filtered and dried in a desiccator under reduced pressureto give the title compound (47.0 g, 76%) of a white solid.

m.p.; 220° C. (dec.), Rf value; 0.58 (CHCl₃:MeOH=10:1).

(d) N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-guanidophenylalanine-N-methylamideMonoacetate

The compound (7.00 g, 7.90 mmol) of Example 8-c was suspended in AcOH(200 ml), added with 5% Pd-C (50% wet, 4.0 g) and then stirredvigorously in a stream of hydrogen at room temperature for 3 hours.After the catalyst was filtered off, AcOH of the filtrate was removedunder reduced pressure. The residue was suspended in AcOEt (300 ml) andstirred for 30 min. The insoluble material was filtered, dried in adesiccator under reduced pressure, added with water (370 ml) andlyophilized, whereby the title compound (4.30 g, 93%) of white amorphouspowder was obtained.

Specific rotation[α]_(D)=−10.2° (c=1.0, MeOH), Rf value; 0.48(CHCl₃:MeOH:AcOH=95:5:3). Analytical value calculated for C₃₀H₄₄N₆O₆,Theoretical value:C, 61.62; H, 7.58; N, 14.37. Found:C, 61.72; H, 7.5;N, 14.12.

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.8 (13H, m, (CH₃)₂CH—CH₂, +CH₂—CH₂ —CH₂—Ph),1.89(3H, s, CH₃ COOH), 2.0-2.8 (7H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph), 2.96(2H, m, C₆H₄ CH₂ ), 4.4-5.0 (1H, m, NH—CH—CO), 6.8-7.5 (9H, m,aromatic-H).

EXAMPLE 9 N-[4-(N-Hydroxyamino)-2(R orS)-isobutylsuccinyl]-O-phospho-L-tyrosine-N-methylamide, Disodium Salt

(a) N-[4-(N-Benzyloxyamino)-2 (R orS)-isobutylsuccinyl]-O-di-tert-butylphospho-L-tyrosine-N-methylamide

The isomer B (2.56 g, 5.62 mmol) of Example 1-e and 1H-tetrazole (1.18g, 16.8 mmol) were dissolved in DMF (25 ml) at room temperature, addedwith di-tert-butyl N,N-diethylphosphoramidite (93% purity, 1.88 g, 7.01mmol) and stirred at room temperature for 2 hours. The reaction mixturewas cooled to −78° C. and dropped with a solution of m-chloroperbenzoicacid (1.80 g, 7.34 mmol) in CH₂Cl₂ (25 ml). The mixture was warmed toroom temperature, stirred for 1 hour, diluted with chloroform (200 ml)and washed with 0.05N-HCl and a saturated aqueous solution of NaHCO₃ twotimes respectively. The organic layer was dried over anhydrous MgSO₄,removed under reduced pressure, added with Et₂O and stirred at roomtemperature for 1 hour. The precipitating solid was filtered and driedin a desiccator under reduced pressure to give the tile compound (2.84g, 80%) of a white solid.

m.p.; 175-177° C., specific rotation[α]_(D)=−17.7° (c=1.0, MeOH), Rfvalue; 0.76 (CHCl₃:MeOH:AcOH=5:2:1), 0.52 (CHCl₃:MeOH=10:1).

¹H-NMR (CDCl₃) δ ppm; 0.82 (6H, m, CH(CH₃)₂ ), 1.49 (9H, s, C(CH₃)₃ ),1.0-2.0(3H, m, (CH₃)₂ CH—CH₂ ), 2.2 (2H, m, CH₂ CO), 2.5-3.5 (6H, m,N—CH₃ +CO—CH₂ CH—CO+CH₂ —C₆H₄), 4.5-5.1 (3H, m, NH—CH—CO+OCH₂ Ph),6.7-7.6 (14H, m, aromatic-H+NH×3).

(b) N-[4-(N-Benzyloxyamino)-2(R orS)-isobutylsuccinyl]-O-phospho-L-tyrosine-N-methylamide, Disodium Salt

To the compound (2.72 g, 4.20 mmol) of Example 9-a was added ice-cooled90% aqueous trifluoroacetic acid (42 ml). The reaction mixture wasstirred at 5° C. for 1 hour and concentrated under reduced pressure. Theresidue was added with Et₂O and stirred at room temperature for 1 hour.The precipitating solid was filtered and dried to give a white solid(1.74 g). The obtained white solid was dissolved in aqueous 1N-NaHCO₃(13 ml) and purified by a reverse-phase column chromatography (FujiSilysia Chemical Ltd., Chromatorex ODS DM-1020T; 100 g, elution with aMeOH-water solution containing 0-20% MeOH) to give the title compound(1.39 g, 57%) of white amorphous powder upon freeze-drying.

Specific rotation[α]_(D)=−11.2° (c=1.0, MeOH), Rf value; 0.08(CHCl₃:MeOH:AcOH=5:2:1), 0.47 (n-BuOH:AcOH:water=4:1:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.8 (9H, m, (CH₃)₂CH—CH₂ ), 2.1 (2H, m, CH₂CO), 2.5-3.2 (6H, m, N—CH₃ +CO—CH₂ CH—CO+CH₂ —C₆H₄), 4.3-5.2 (3H, m,NH—CH—CO+OCH₂ Ph), 6.9-7.6 (9H, m, aromatic-H).

(c) N-[4-(N-Hydroxyamino)-2(R orS)-isobutylsuccinyl]-O-phospho-L-tyrosine-N-methylamide, Disodium Salt

The compound (1.26 g, 2.17 mmol) of Example 9-b was dissolved in MeOH(50 ml), added with 5% Pd-C (50% wet, 1.26 g), and then stirredvigorously in a stream of hydrogen at room temperature for 4 hours.After the catalyst was filtered off, MeOH of the filtrate was removedunder reduced pressure. The residue was suspended in water (50 ml) andstirred for 30 min. The insoluble material was filtered and the filtratewas lyophilized, whereby the title compound (790 mg, 75%) of whiteamorphous powder was obtained.

Specific rotation[α]_(D)=+10.6° (c=1.0, water), Rf value; 0.31(n-BuOH:AcOH:water=4:1:1). Analytical value calculated forC₁₈H₂₆N₃O₈Na₂P. Theoretical value:C, 44.18; H, 5.36; N, 8.59. Found:C,43.91; H, 5.48; N, 8.55.

¹H-NMR(MeOH-d₄+D₂O) δ ppm; 0.5-1.8 (9H, m, (CH₃)₂CH—CH₂ ), 2.1 (2H, m,CH₂ CO), 2.4-3.2 (6H, m, N—CH₃ +CO—CH₂ CH—CO+CH₂ —C₆H₄), 4.3-4.8 (1H, m,NH—CH—CO), 7.15 (4H , s, aromatic-H).

EXAMPLE 10 N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-acetimidoyliminomethylenephenylalanine-N-methylamideMonoacetate

(a) N-[4-tert-Butoxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-cyanophenylalanine-N-methylamide

The compound (3.62 g, 10.4 mmol) of Example 3-d,L-4′-cyanophenylalanine-N-methylamide hydrochloride (3.62 g, 10.4 mmol)and HOBT (1.55 g, 11.5 mmol) were dissolved in DMF (70 ml) and addedwith TEA (1.40 ml, 12.5 mmol) and EDC (2.40 g, 12.5 mmol) at −15° C.under stirring. The mixture was stirred at −15° C. for 1 hour andfurther at 5° C. overnight and removed under reduced pressure. Theresidue was dissolved in AcOEt (200 ml), and washed with a saturatedaqueous solution of NaCl, 1N-HCl, a saturated aqueous solution of NaHCO₃and then the saturated aqueous solution of NaCl two times respectively.The organic layer was dried over anhydrous MgSO₄₁ evaporated underreduced pressure to remove the solvent, purified by columnchromatography (silica gel; 1 Kg, elution with the mixed solvent;CHCl₃MeOH=50:1) and recrystallized from AcOEt-n-hexane to give the titlecompound (1.71 g, 31%) of a white solid.

m.p.; 196-198° C., specific rotation[α]_(D)=−13.3° (c=1.1, MeOH), Rfvalue; 0.65 (CHCl₃:MeOH=10:1), 0.52 (CHCl₃:MeOH:AcOH=95:5:3).

¹H-NMR(CDCl₃) δ ppm; 0.81 (6H, m, CH(CH₃)₂ ), 0.9-1.8 (16H, s+m, (CH₃)₂CH—CH₂, +C(CH₃)₃ +CH₂—CH₂ —CH₂—Ph), 2.1-2.85 (7H, d+m, J=4.6 Hz, N—CH₃+—CH—CO×2+CH₂ —Ph), 3.02 (2H, m, C₆H₄ CH₂ ), 4.62 (1H, m, NH—CH—CO),6.10 (1H, m, NH), 6.50 (1H, m, NH), 6.9-7.7 (9H, m, aromatic-H).

(b) N-[4-Hydroxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-cyanophenylalanine-N-methylamide

To the compound (1.73 g, 3.24 mmol) of Example 10-a was added ice-cooled95% aqueous trifluoroacetic acid (16 ml). The reaction mixture wasstirred at 5° C. for 4 hours and concentrated under reduced pressure.The concentrate was added with Et₂O and stirred at room temperature for1 hour. The precipitating solid was filtered and dried to give the titlecompound (1.22 g, 79%) of a white solid.

m.p.; 234-235° C., specific rotation[α]_(D)=−8.7° (c=1.1, MeOH), Rfvalue; 0.35 (CHCl₃:MeOH=10:1), 0.46(CHCl₃:MeOH:AcOH=95.5:3).

¹H-NMR(MeOH-d₄) δ ppm; 0.83 (6H, m, CH(CH₃)₂ ), 0.95-1.85 (7H, m, (CH₃)₂CH—CH₂, +CH₂—CH₂ —CH₂—Ph), 2.2-3.2 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄CH₂ ), 4.60 (1H, m, NH—CH—CO), 6.9-7.25 (9H, m, aromatic-H).

(c) N-[4-Hydroxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-aminomethylenephenylalanine-N-methylamideHydrochloride

The compound (1.22 g, 2.55 mmol) of Example 10-b was dissolved inethanol (25 ml), added with conc. HCl (1 ml) and 5% Pd-C (50% wet, 600mg) and then stirred vigorously in a stream of hydrogen at roomtemperature for 10 hours. After the catalyst was filtered off, ethanolof the filtrate was removed under reduced pressure. The residue wassuspended in water (50 ml) and stirred for 30 min. The insolublematerial was taken by filtration and dried in a desiccator under reducedpressure, added with Et₂O, and stirred at room temperature for 1 hour.The precipitating solid was filtered and dried in the desiccator underreduced pressure to give the title compound (1.15 g, 94%) of a whitesolid.

m.p.; 194-202° C., specific rotation[α]_(D)=−15.1° (c=1.0, MeOH), Rfvalue; 0.32 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.6-0.95 (6H, m, CH(CH₃)₂ ), 1.0-1.8 (7H, m,(CH₃)₂ CH—CH₂, +CH₂—CH₂ —CH₂—Ph), 2.2-2.7 (7H, m, N—CH₃ +—CH—CO×2+CH₂—Ph), 2.95 (2H, m, C₆H₄ CH₂ ), 4.0 (2H, brs, CH₂ —NH₂), 4.63 (1H, m,NH—CH—CO), 6.9-7.6 (9H, m, aromatic-H).

(d) N-[4-Hydroxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-acetimidoyliminomethylenephenylalanine-N-methylamideHydrochloride

The compound (530 mg, 1.02 mmol) of Example 10-c was dissolved in DMF(10 ml), added with ethyl acetimidate hydrochloride (252 mg, 2.04 mmol)and TEA (416 μl, 3.00 mmol) under stirring in an ice bath and stirredfor 1 hour. The DMF was removed under reduced pressure. The residue wasadded with 1N-HCl to adjust pH to 2, and then purified by DIAION HP-20(Mitsubishi Chemicals; 100 ml, elution with a MeOH-water solutioncontaining 10-80% MeOH), whereby water (20 ml) was added to give thetitle compound (511 mg, 65%) of white amorphous powder uponfreeze-drying.

Specific rotation[α]_(D)=−16.50° (c=1.0, MeOH), Rf value; 0.23(CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.0 (6H, m, CH(CH₃)₂ ), 1.0-1.8 (7H, m,(CH₃)₂ CH—CH₂, +CH₂—CH₂ —CH₂—Ph), 2.17 (3H, s, C—CH₃ ), 2.26-2.77 (7H,m, N—CH₃ +—CH—CO×2+CH₂ —Ph), 2.8-3.2 (2H, m, C₆H₄ CH₂ ), 4.38 (2H, brs,CH₂ —NH), 4.58(1H, m, NH—CH—CO), 6.8-7.55 (9H, m, aromatic-H).

(e) N-[4-(N-Bezyloxyamino-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-acetimidoyliminomethylenephenylalanine-N-methylamideHydrochloride

The compound (465 mg, 0.832 mmol) of Example 10-d, O-benzylhydroxylaminehydrochloride (200 mg, 1.25 mmol) and HOBT (169 mg, 1.25 mmol) weresuspended in DMF (10 ml). The suspension was added with TEA (288 μl,2.08 mmol) and EDC (240 mg, 1.25 mmol) at −15° C. under stirring. Themixture was stirred at −15° C. for 1 hour and further at roomtemperature overnight. The DMF was removed under reduced pressure. Theresidue was added with 1N-HCl to adjust pH to 2 and then purified byDIAION HP-20 (Mitsubishi Chemicals; 100 ml, elution with a MeOH-watersolution containing 20-80% MeOH) and column chromatography (silica gel;50 g, elution with the mixed solvent; CHCl₃:MeOH=9:1-1:1, MeOH andAcOH), whereby water (20 ml) was added to give the title compound (420mg, 76%) of white amorphous powder upon freeze-drying.

Specific rotation[α]_(D)=−1.3° (c=1.0, MeOH), Rf value; 0.25(CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.7(13H, m, (CH₃)₂CH—CH₂, +CH₂—CH₂ —CH₂—Ph),1.90(3H, s, CH₃ COOH), 2.0-3.1 (12H, s+m, C—CH₃ +N—CH₃ +—CH—CO×2+CH₂—Ph+C₆H₄ CH₂ ), 4.36 (2H, brs, CH₂ —NH), 4.4-5.2 (3H, s+m, NH—CH—CO+OCH₂Ph), 6.8-7.6 (14H, m, aromatic-H).

(f) N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-acetimidoyliminomethylenephenylalanine-N-methylamideMonoacetate

The compound (200 mg, 0.291 mmol) of Example 10-e was dissolved in AcOH(10 ml), added with 5% Pd-C (50% wet, 200 mg), and then stirredvigorously in a stream of hydrogen at room temperature for 30 hours.After the catalyst was filtered off, AcOH of the filtrate was removedunder reduced pressure. The residue was added with water (10 ml) to givethe title compound (159 mg, 91%) of a white amorphous powder uponfreeze-drying.

Specific rotation[α]_(D)=−8.9° (c=1.0, MeOH), Rf value; 0.13(CHCl₃:MeOH:AcOH=5:2:1), 0.50(n-BuOH:AcOH:water=4:1:1). Analytical valuecalculated for C₃₂H₄₇N₅O₆. Theoretical value:C, 64.3; H, 7.93; N, 11.72.Found:C, 64.35; H, 8.01; N, 11.68.

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.7 (13H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph),1.90(3H, s, CH₃ COOH), 2.0-3.1 (12H, s+m, C—CH₃ +N—CH₃ +—CH—CO×2+CH₂—Ph+C₆H₄ CH₂ ), 4.36 (2H, brs, CH₂ —NH), 4.6 (1H, m, NH—CH—CO), 6.8-7.6(9H, m, aromatic-H).

EXAMPLE 11 N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′propionimidoyliminomethylenephenylalanine-N-methylamideMonoacetate

By the same methods as used in d-f of Example 10, the followingcompounds were synthesized from the compound of Example 10-c and ethylpropionimidate hydrochloride.

(a) N-[4-Hydroxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-propionimidoyliminomethylenephenylalanine-N-methylamideHydrochloride

White amorphous powder (79%), specific rotation[α]_(D)=−14.1° (c=0.2,MeOH), Rf value; 0.32 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.0 (6H, m, CH(CH₃)₂ ), 1.0-1.8 (10H, m,(CH₃)₂ CH—CH₂, +CH₂—CH₂ —CH₂—Ph+CH₂—CH₃ ), 2.2-2.75 (9H, m, N—CH₃+—CH—CO×2+CH₂ —Ph+CH₂ —CH₃), 2.95 (2H, m, C₆H₄ CH₂ ), 4.38 (2H, brs, CH₂—NH), 4.59 (1H, m, NH—CH—CO), 6.9-7.5 (9H, m, aromatic-H).

(b) N-[4-(N-Bezyloxyamino-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-propionimidoyliminomethylenephenylalanine-N-methylamideHydrochloride

White amorphous powder (76%), specific rotation[α]_(D)=−4.4° (c=1.0,MeOH), Rf value; 0.53 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.55-1.0 (6H, m, CH(CH₃)₂ ), 1.0-1.75 (10H, m,(CH₃)₂ CH—CH₂ ,+CH₂—CH₂ —CH₂—Ph+CH₂—CH₃ ), 2.2-2.8 (9H, m, N—CH₃+—CH—CO×2+CH₂ —Ph+CH₂ —CH₃), 2.95(2H, m, C₆H₄ CH₂ ), 4.4-4.75 (3H, m CH₂—NH+NH—CH—CO), 4.84 (2H, s, OCH₂ Ph), 6.9-7.6 (14H, m, aromatic-H).

(c) N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-propionimidoyliminomethylenephenylalanine-N-methylamideMonoacetate

White amorphous powder (91%), specific rotation[α]_(D)=−9.7° (c=0.2,MeOH), Rf value; 0.35 (CHCl₃:MeOH:AcOH=5:2:1). Analytical valuecalculated for C₃₃H₄₉N₅O₆. Theoretical value:C, 64.79; H, 8.07; N,11.45. Found:C, 64.95; H, 8.12; N, 11.38.

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.75 (16H, m, (CH₃)₂CH—CH₂, +CH₂—CH₂—CH₂—Ph+CH₂—CH₃ ), 1.88 (3H, s, CH₃ COOH), 2.2-3.2 (11H, m, N—CH₃+—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ +CH₂ —CH₃), 4.38 (2H, brs, CH₂ —NH), 4.45-4.8(1H, m, NH—CH—CO), 6.9-7.5 (9H, m, aromatic-H).

EXAMPLE 12N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-benzimidoyliminomethylenephenylalanine-N-methylamideMonoacetate

By the same methods as used in d-f of Example 10, the followingcompounds were synthesized from the compound (3-RS type) of Example 10-cand ethyl benzimidate hydrochloride.

(a)N-[4-Hydroxy-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-benzimidoyliminomethylenephenylalanine-N-methylamideHydrochloride

White amorphous powder (71%), specific rotation[α]_(D)=−9.0° (c=1.0,MeOH), Rf value; 0.34 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.4-0.95 (6H, m, CH(CH₃₎ ₂ , 0.95-1.8 (7H, m,(CH₃)₂ CH—CH₂, +CH₂—CH₂ —CH₂—Ph), 2.1-2.8 (7H, m, N—CH₃ +—CH—CO×2+CH₂—Ph), 2.95 (2H, m, C₆H₄ CH₂ ), 3.6-4.6 (4H, m, CH₂ —NH+NH—CH—CO),6.9-8.0 (14H, m, aromatic-H).

(b)N-[4-(N-Bezyloxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-benzimidoyliminomethylenephenylalanine-N-methylamideHydrochloride

White amorphous powder (92%), specific rotation[α]_(D)=−4.6° (c=1.1,MeOH), Rf value; 0.41 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.0 (6H, m, CH(CH₃)₂ ), 1.0-1.8 (7H, m,(CH₃)₂ CH—CH₂, +CH₂—CH₂ —CH₂—Ph), 1.8-3.3 (9H, m, N—CH₃ +—CH—CO×2+CH₂—Ph+C₆H₄ CH₂ ), 4.4-5.1 (5H, m, CH₂ —NH+NH—CH—CO+OCH₂ Ph), 6.9-8.1 (19H,m, aromatic-H).

(c)N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-benzimidoyliminomethylenephenylalanine-N-methylamideMonoacetate

White amorphous powder (79%), specific rotation[α]_(D)=−4.3° (c=1.1,MeOH), Rf value; 0.54 (CHCl₃:MeOH:ACOH=5:2:1). Analytical valuecalculated for CH₃₇H₄N₅O₆. Theoretical value:C, 67.35; H, 1.49; N,10.61. Found:C, 67.6; H, 7.34; N, 10.44.

¹H-NMR(MeOH-d₄) δ ppm; 0.55-1.85 (13H, m, (CH₃)₂CH—CH₂, +CH₂—CH₂—CH₂—Ph), 1.93 (3H, s, CH₃ COOH), 2.0-3.4 (9H, m, N—CH₃ +—CH—CO×2+CH₂—Ph+C₆H₄ CH₂ ), 4.4-4.8 (3H, m, CH₂ —NH+NH—CH—CO), 7.0-7.9 (14H, m,aromatic-H).

EXAMPLE 13N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-acetamidomethylenephenylalanine-N-methylamide

(a)N-[4-Hydroxy-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-acetamidomethylenephenylalanine-N-methylamide

The compound (3-RS type, 500 mg, 0.97 mmol) of Example 10-c wasdissolved in DMF (10 ml), added with acetic anhydride (109 μl, 1.16mmol) and TEA (285 μl, 2.04 mmol) under stirring in an ice bath andstirred for 1 hour. The DMF was removed under reduced pressure. Theresidue was added with Et₂O and stirred at room temperature for 1 hour.The precipitating solid was filtered and dried in a desiccator underreduced pressure to give the title compound (500 mg, 98%) of a whitesolid.

m.p.; 155-161° C., specific rotation[α]_(D)=−14.3° (c=1.04, DMF), Rfvalue; 0.24 (CHCl₃:MeOH=10:1).

¹H-NMR(DMSO-d₄) δ ppm; 0.3-1.65 (13H, m, (CH₃)₂CH—CH₂, +CH₂—CH₂—CH₂—Ph), 1.80+1.82 (3H, s each, CH₃ —CO), 1.9-3.7 (9H, m, N—CH₃+—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ ), 4.0-4.7 (3H, m, CH₂ —NH+NH—CH—CO), 6.9-8.5(12H, m, aromatic-H+NH×3).

(b)N-[4-(N-Bezyloxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-acetamidomethylenephenylalanine-N-methylamide

By the same method (however, the DIAION HP-20 treatment was not done) asthat in Example 10-e, the title compound (520 mg, 96%) of a white solidwas obtained from the compound (450 mg, 0.86 mmol) of

EXAMPLE 13-a

m.p.; 233-239° C. Rf value; 0.36 (CHCl₃:MeOH=10:1).

¹H-NMR(DMSO-d₄) δ ppm; 0.4-1.0 (6H, m, CH(CH₃)₂ ), 1.0-1.6 (7H, m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.80+1.82 (3H, s each, CH₃ —CO),1.9-3.2 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ ), 4.0-4.7 (3H, m, CH₂—NH+NH—CH—CO), 4.76 (2H, s, OCH₂ Ph), 6.8-8.5 (18H, m, aromatic-H+NH×4).

(c)N-[4-(N-Hydroxyamino-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-acetamidomethylenephenylalanine-N-methylamide

By the same method as that in Example 10-f, the title compound (130 mg,32%) of white amorphous powder was obtained from the compound (470 mg,0.75 mmol) of Example 13-b.

Specific rotation[α]_(D)=−15.0° (c=0.32, DMF), Rf value; 0.26(CHCl₃:MeOH=10:1), 0.34 (CHCl₃:MeOH:AcOH=5:2:1). Analytical valuecalculated for C₃₀H₄₂N₄O₅. Theoretical value:C, 66.89; H, 7.86; N, 10.4.Found:C, 66.85; H, 7.87; N, 10.44.

¹H-NMR (DMSO-d₄) δ ppm; 0.3-1.65 (13H, m, (CH₃)₂CH—CH₂, +CH₂—CH₂—CH₂—Ph), 1.80+1.82 (3H, s each, CH₃ —CO), 1.9-3.7 (9H, m, N—CH₃+—CH—CO×2+CH₂ —Ph+C₆H₄ CH₂ ), 4.0-4.7 (3H, m, CH₂ —NH+NH—CH—CO), 6.9-8.5(13H, m, aromatic-H+NH×4).

EXAMPLE 14N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-guanidomethylenephenylalanine-N-methylamideMonoacetate

(a)N-[4-Hydroxy-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-[N,N′-bis(benzyloxycarbonyl)guanidomethylene]phenylalanine-N-methylamideMonoacetate

The compound (3-RS type, 500 mg, 0.97 mmol) of Example 10-c wasdissolved in DMF (8 ml), added with1H-pyrazole-N,N′-bis(benzyloxycarbonyl) carboxamidine (470 mg, 1.16mmol) and TEA (162 μl, 1.16 mmol) under stirring in an ice bath andstirred at room temperature overnight. The DMF was removed under reducedpressure. The residue was added with Et₂O and stirred at roomtemperature for 1 hour. The precipitating solid was filtered and driedin a desiccator under reduced pressure to give the title compound (650mg, 86%) of a white solid.

m.p.; 141-145° C., specific rotation[α]_(D)=−14.0° (c=0.71, DMF), RPvalue; 0.41 (CHCl₃:MeOH=10:1).

¹H-NMR(DMSO-d₆) δ ppm; 0.3-0.9 (6H, m, CH(CH₃)₂ ), 0.9-1.8 (7H, m,(CH₃)₂ CH—CH₂, +CH₂—CH₂ —CH₂—Ph), 2.0-3.1 (9H, m, —CH—CO×2+CH₂ —Ph+N—CH₃+C₆H₄ CH₂ ), 4.3-4.7 (3H, m, CH₂ —NH+NH—CH—CO), 5.03 (2H, s, OCH₂ Ph),5.19 (2H, s, OCH₂ Ph), 6.8-7.6 (20H, m, aromatic-H+NH), 7.8-8.7 (1H, m,NH×3).

(b)N-[4-(N-benzyloxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-[N,N′-bis(benzyloxycarbonyl)guanidomethylene]phenylalanine-N-methylamide

The compound (600 mg, 0.76 mmol) of Example 14-a, O-benzylhydroxylaminehydrochloride (180 mg, 1.14 mmol) and HOBT (120 mg, 0.91 mmol) weresuspended in DMF (8 ml). The suspension was added with TEA (160 μl, 1.14mmol) and EDC (170 mg, 0.91 mmol) at −15° C. under stirring. The mixturewas stirred at −15° C. for 1 hour and further at room temperatureovernight. The reaction mixture was dropped into 0.5N-HCl. Theprecipitating crystals were filtered, washed with aqueous 10% Na₂CO₃,water and then Et₂O and dried in a desiccator under reduced pressure togive the title compound (520 mg, 76%) of a white solid.

m.p.; 233-237° C., Rf value; 0.57 (CHCl₃:MeOH=10:1).

¹H-NMR(DMSO-d₆) δ ppm; 0.35-0.9 (6H, m, CH(CH₃)₂ ), 0.9-1.8 (7H, m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.8-3.0 (9H, m, —CH—CO×2+CH₂ —Ph+N—CH₃+C₆H₄ CH₂ ), 4.25-4.6 (3H, m, CH₂ —NH+NH—CH—CO), 4.76 (2H, s, OCH₂ Ph),5.02 (2H, s, OCH₂ Ph), 5.19 (2H, s, OCH₂ Ph), 6.8-7.5 (26H, m,aromatic-H+NH×2), 7.8-8.7 (1H, m, NH×3).

(c)N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-guanidomethylenephenylalanine-N-methylamideMonoacetate

The compound (400 mg, 0.45 mmol) of Example 14-b was suspended in AcOH(20 ml), added with 5% Pd-C (50% wet, 400 mg) and then stirredvigorously in a stream of hydrogen at room temperature for 4 hours.After the catalyst was filtered off, AcOH of the filtrate was removedunder reduced pressure. The residue was added with water (20 ml) to givethe title compound (240 mg, 90%) of white amorphous powder uponfreeze-drying.

Specific rotation[α]_(D)=−9.34° (c=0.97, DMF), Rf value; 0.19(CHCl₃:MeOH:AcOH=5:2:1). Analytical value calculated for C₃₁H₄₆N₆O₆.Theoretical value:C, 62.19; H, 7.74; N, 14.04. Found:C, 62.2; H, 7.67;N, 14.04.

¹H-NMR(DMSO-d₆) δ ppm; 0.4-0.9 (6H, m, CH(CH₃)₂ ), 1.05-1.55 (7H, m,(CH₃)₂ CH—CH₂, +CH₂—CH₂ —CH₂—Ph), 1.70 (3H, s, CH₃ COOH), 2.1-3.0 (9H,m, —CH—CO×2+CH₂ —Ph+N—CH₃ +C₆H₄ CH₂ ), 4.0-4.6 (3H, m, CH₂—NH+NH—CH—CO), 6.9-7.5 (9H, m, aromatic-H+NH×2), 7.7-8.4 (4H, m, NH×4).

EXAMPLE 15 N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-meta-tyrosine-N-methylamide,Monosodium Salt

By the same methods as used in e-i oi Example 3, the following compoundswere synthesized from L-meta-tyrosine-N-methylamide hydrochlodide.

(a) N-[4-tert-Butoxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-meta-tyrosine-N-methylamide.

White solid (26%), m.p.; 72° C., specific rotation[α]_(D)=−15.7° (c=1.0,CHCl₃), Rf value; 0.57 (CHCl₃:MeOH=10:1).

¹H-NMR(CDCl₃) δ ppm; 0.82 (6H, m, CH(CH₃)₂ ), 0.9-2.0 (16H, s+m, (CH₃)₂CH—CH₂ +C(CH₃)₃ +CH₂—CH₂ —CH₂—Ph), 2.2-3.2 (9H, m, N—CH₃ +—CH—CO×2+CH₂—Ph+C₆H₄ CH₂ ), 4.72 (1H, m, NH—CH—CO), 6.3-7.4 (11H, m,aromatic-H+NH×2), 8.00 (1H, brs, OH).

(b) N-[4-Benzyloxyamino-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-meta-tyrosine-N-methylamide

White solid (62%), m.p.; 229° C., specific rotation[α]_(D)=−8.8° (c=1.0,MeOH), Rf value; 0.48 (CHCl₃:MeOH=10:1).

¹H-NMR (MeOH-d₄) δ ppm; 0.8 (6H, m, CH(CH₃)₂ ), 0.9-1.7 (7H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.8-3.2 (9H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph+C₆H₄CH₂ ), 4.4-4.8 (1H, m, NH—CH—CO), 4.82 (2H, s, OCH₂ Ph), 6.5-7.6 (14H,m, aromatic-H).

(c) N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-meta-tyrosine-N-methylamide,Monosodium Salt

White amorphous powder (71%), specific rotation[α]_(D)=+2.6° (c=1.0,MeOH), Rf value; 0.66 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.6-1.0 (6H, m, CH(CH₃)₂ ), 1.0-1.7 (7H, m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.8-3.2 (9H, m, N—CH₃ 30 —CH—CO×2+CH₂—Ph+C₆H₄ CH₂ ), 4.4-4.7 (1H, m, NH—CH—CO), 4.82 (s, OCH₂ Ph), 6.9-7.6(14H, m, aromatic-H).

(d) N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-meta-tyrosine-N-methylamide,Monosodium Salt

White amorphous powder (quantative yield), specificrotation[α]_(D)=−3.7° (c=1.0, MeOH), Rf value; 0.49(CHCl₃:MeOH:AcOH=5:2:1). Analytical value calculated for C₂₇H₃₆N₃O₈NaS.Theoretical value:C, 55.37; H, 6.2; N, 7.17. Found:C, 55.22; H, 6.04; N,7.03.

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.75 (13H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph),1.9-2.85 (7H, m, N—CH₃ +—CH—CO×2+CH₂ —Ph), 2.9 (2H, m, C₆H₄ CH₂ ), 4.65(1H, m, NH—CH—CO), 6.8-7.4 (9H, m, aromatic-H).

EXAMPLE 16N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-hydroxysuccinyl]-L-4′-guanidophenylalanine-N-methylamideMonoacetate

(a) Methyl 3(R)-carboxy-2(S)-hydroxy-5-methylhexanoate

3(R)-Carboxy-2(S)-hydroxy-5-methylhexanoate (440 mg, 2.31 mmol) wasadded with trifluoroacelic anhydrode (4 ml) and stirred at 0° C. for 4hours. The mixture was evaporated under reduced pressure. The residuewas added with MeOH (4 ml), stirred at 0° C. for 2 hours, evaporatedunder reduced pressure to remove MeOH and purified by columnchromatography (silica gel; 35 g, elution with the mixed solvent;CHCl₃:MeOH=20:1) to give the title compound (344 mg, 73%) of a colorlessoil.

¹H-NMR(CDCl₃) δ ppm; 0.94 (6H, d, J=5.0 Hz, CH(CH₃)₂ ), 1.3-2.0 (3H, m,(CH₃)₂ CH—CH₂,), 2.8-3.2 (1H, m CH—CO₂H), 3.82 (3H, s, OCH₃), 4.29 (1H,d, J=3.5 Hz, HO—CH), 6.6 (2H, brm, OH+CO₂H).

(b)N-[3(S)-Hydroxy-2(R)-isobutyl-4-methoxysuccinyl]-L-4′-[N,N′-bis(benzyloxycarbonyl)guanido]phenylalanine-N-methylamide

The compound (270 mg, 1.80 mmol) of Example 16-a,L-4′-[N,N′-bis(benzyloxycarbonyl)guanido]phenylalanine-N-methylamidehydrochloride (1.19 g, 2.20 mmol) and HOBT (540 mg, 4.00 mmol) weredissolved in DMF (6 ml). The solution was added with TEA (620 μl, 4.40mmol) and EDC (420 mg, 2.20 mmol) at −15° C. under stirring. The mixturewas stirred at −15° C. for 1 hour and further at 5° C. overnight andevaporated under reduced pressure. The residue was added with AcOEt (30ml) and washed with a saturated aqueous solution of NaCl, 1N-HCl, asaturated aqueous solution of NaHCO₃ and then the saturated aqueoussolution of NaCl two times respectively. The organic layer was driedover anhydrous MgSO₄, evaporated under reduced pressure and purified bycolumn chromatography (silica gel; 50 g, elution with the mixed solvent;CHCl₃:MeOH=50:1) followed by crystallizing from chloroform-n-hexane togive the title compound (0.40 g, 45%) of a white solid.

m.p.; 175° C., specific rotation[α]_(D)=−17.4° (c=1.03, CHCl₃), Rfvalue; 0.20 (CHCl₃:MeOH=50:1).

¹H-NMR(CDCl₃) δ ppm; 0.7-1.1 (6H, m, CH(CH₃)₂ ), 1.0-1.6 (3H, m, (CH₃)₂CHCH₂ ), 2.4-3.5 (6H, d+m, J=4.8 Hz, N—CH₃ +CH—CO+CH₂ —C₆H₄), 3.65 (3H,s, CO₂ CH₃ ), 3.90 (1H, m, NH), 4.23 (1H, m, NH—CH—CO), 4.4-4.7 (1H, m,HO—CH—CO), 5.08 (2H, s, Ph—CH₂ —O), 5.25 (2H, s, Ph—CH₂ —O), 6.34 (1H,m, NH), 6.75 (1H, m, HO), 7.0-7.7 (16H, m, aromatic-H+NH×2).

(c)N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-hydroxysuccinyl]-L-4′-guanidophenylalanine-N-methylamideMonoacetate

The compound (0.32 g, 0.46 mmol) of Example 16-b was added an ice-cooledMeOH solution of alkaline hydroxylamine (0.7 M, 5.0 ml), and the mixturewas stirred at room temperature for 3 hours. The reaction liquid wasmade pH 3-4 by 1N-HCl and evaporated under reduced pressure.

The residue was dissolved in AcOH (20 ml), added with 5% Pd-C (50% wet,0.5 g) and then stirred vigorously in a stream of hydrogen at roomtemperature for 5 hours. After the catalyst was filtered off, AcOH ofthe filtrate was removed under reduced pressure. The residue waspurified by a reverse-phase column chromatography (Fuji Silysia ChemicalLtd., Chromatorex ODS DM-1020T; 50 g, elution with a MeOH-water solutioncontaining 0-20% MeOH) to give the tille compound (0.12 g, 54%) of whiteamorphous powder upon freeze-drying.

Specific rotation[α]_(D)=+8.27° (c=1.04, MeOH), Rf value; 0.22(CHCl₃:MeOH:AcOH=5:2:1). Analytical value calculated for C₂₁H₃₄N₆O₇.Theoretical value:C, 52.27; H, 7.1; N, 17.42. Found:C, 52.32; H, 7.21;N, 17.35.

¹H-NMR(MeOH-d₄) δ ppm; 0.86 (6H, m, CH(CH₃)₂ ), 1.0-1.85 (3H, m, (CH₃)₂CHCH₂ ), 1.90 (3H, s, CH₃ CO₂H), 2.5-2.85 (4H, m, N—CH₃ +CH—CO), 2.9-3.2(2H, m, CH₂ —C₆H₄), 4.5-5.0 (2H, m, NH—CH—CO+HO—CH—CO), 7.0-7.55 (4H, m,aromatic-H).

EXAMPLE 17 N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-amidinophenylalanine-N-methylamideMonoacetate

By the same methods as used in b-d of Example 8, the following compoundswere synthesized fromN^(α)-tert-butyloxycarbonyl-4′-[N-(benzyloxycarbonyl)amidino]phenylalanine-N-methylamide.

(a) N-[4-tert-Butoxy-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-[N-(benzyloxycarbonyl)amidino]phenylalanine-N-methylamide

White solid (18%), m.p.; 233° C. (dec.), specific rotation[α]_(D)=+0.99°(c=1.1, MeOH), Rf value; 0.48 (CHCl₃:MeOH=10:1).

¹H-NMR(CDCl₃) δ ppm; 0.6-1.0 (6H, m, (CH₃)₂ CH—CH₂), 1.0-1.6 (16H, s+m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph+C(CH₃)₃ ), 2.2-2.8 (7H, d+m, J=4.7 Hz,—CH—CO×2+CH—Ph+N—CH₃ ), 3.10 (2H, m, C₆H₄ CH₂ ), 4.56 (1H, m, NH—CH—CO),5.20 (2H, s, Ph—CH₂ —O), 6.2-6.6 (2H, m, NH×2), 7.1-7.5 (14H, m,aromatic-H), 7.7-7.9 (2H, m, NH×2).

(b) N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-[N-(benzyloxycarbonyl)amidino]phenylalanine-N-methylamide

White solid (38%), m.p.; 259-261° C., Rf value; 0.40 (CHCl₃:MeOH=10:1).

¹H-NMR(CDCl₃) δ ppm; 0.85 (6H, m, (CH₃)₂ CH—CH₂), 1.2-2.1 (9H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph+—CH—CO×2), 2.5-3.2 (7H, m, C₆H₄ CH₂ +CH₂—Ph+N—CH₃ ), 4.5-5.1 (5H, m, NH—CH—CO+Ph—CH₂ —O×2), 6.9-7.7 (19H, m,aromatic-H).

(c) N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(R orS)-(3-phenyltrimethylene)succinyl]-L-4′-amidinophenylalanine-N-methylamideMonoacetate

White amorphous powder (96%), specific rotation[α]_(D)=−7.04° (c=1.0,MeOH), Rf value; 0.45 (CHCl₃:MeOH:AcOH=5:2:1). Analytical valuecalculated for C₃₀H₄₃N₅O₆. Theoretical value:C, 63.25; H, 7.61; N,12.29. Found:C, 63.33; H, 7.62; N, 12.34.

¹H-NMR(MeOH-d₄) δ ppm; 0.85 (6H, m, (CH₃)₂ CH—CH₂), 1.1-1.75 (7H, m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.91 (3H, s, CH₃ CO₂H), 2.0-2.55 (4H,m, —CH—CO×2+CH₂ —Ph), 2.67 (3H, m, N—CH₃ ), 2.8-3.2 (2H, m, C₆H₄ CH₂ ),4.5-5.0 (1H, m, NH—CH—CO), 6.9-7.85 (9H, m, aromatic-H).

EXAMPLE 18N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-1-naphthylamide,Monosodium Salt

By the same methods as used in e-i of Example 3, the following compoundswere synthesized from L-tyrosine-N-1-naphthylamide hydrochloride.

(a)N-[4-tert-Butoxy-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-tyrosine-N-1-naphthylamide

m.p.; 89-93° C., specific rotation[α]_(D)=−18.8° (c=0.97, CHCl₃), Rfvalue; 0.72 (n-hexane:AcOEt=1.1).

¹H-NMR(CDCl₃) δ ppm; 0.6-0.9 (6H, m, (CH₃)₂ CH—CH₂), 0.9-1.85 (16H, m,(CH₃)₂ CH—CH₂ +CH—CH₂—CH₂ —Ph+C(CH₃)₃ ), 2.2-2.6 (4H, m, CH—CO×2+Ph—CH₂), 3.1 (2H, m, CH₂ —C₆H₄), 4.97 (1H, m, NH—CH—CO), 6.6-8.1 (16H, m,aromatic-H).

(b)N-[4-Benzyloxyamino-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-tyrosine-N-1-naphthylamide

m.p.; 210-235° C., specific rotation[α]_(D)=−14.2° (c=1.0, DMF), Rfvalue; 0.58 (CHCl₃:MeOH=10:1).

¹H-NMR(DMSO-d₆) δ ppm; 0.4-0.95 (6H, m, (CH₃)₂ CH—CH₂), 1.0-1.6 (7H, m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.8-2.6 (4H, m, CH—CO×2+Ph—CH₂ ), 2.95(2H, m, CH₂ —C₆H₄), 4.6-5.0 (3H, m, NH—CH—CO+Ph—CH₂ —O), 6.4-8.1 (21H,m, aromatic-H).

(c)N-[4-(N-Benzyloxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-1-naphthylamide,Monosodium Salt

White amorphous powder (87%), specific rotation[α]_(D)=−15.7° (c=1.0,MeOH), Rf value; 0.62 (CHCl₃:MeOH:AcOH=5:2:1).

¹H-NMR(MeOH-d₄) δ ppm; 0.6-1.0 (6H, m, (CH₃)₂ CH—CH₂), 1.0-1.8 (7H, m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.9-2.8 (4H, m, CH—CO×2+Ph—CH₂ ),2.9-3.2 (2H, m, CH₂ —C₆H₄), 4.5-5.1 (3H, s+m, NH—CH—CO+Ph—CH₂ —O), 6.75(1H, m, NH), 6.9-8.0 (21H, m, aromatic-H).

(d)N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-O-sulfo-L-tyrosine-N-1-naphthylamide,Monosodium Salt

White amorphous powder (94%), Rf value; 0.24 (CHCl₃:MeOH:AcOH=5:2:1).Analytical value calculated for C₃₆H₄₀N₃O₈NaS. Theoretical value:C,61.97; H, 5.78; N, 6.02. Found:C, 61.74; H, 5.66; N, 5.97.

¹H-NMR(MeOH-d₄) δ ppm; 0.5-1.0 (6H, m, (CH₃)₂ CH—CH₂), 1.0-1.8 (7H, m,(CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.8-2.8 (4H, m, CH—CO×2+Ph—CH₂ ), 3.1(2H, m, CH₂ —C₆H₄), 4.6-5.2 (1H, m, NH—CH—CO), 6.6-8.0 (16H, m,aromatic-H).

EXAMPLE 19N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-guanidophenylalanine-N-phenylamideMonoacetate

By the same methods as used in b-d of Example 8, the following compoundswere synthesized fromN^(α)-tert-butyloxycarbonyl-4′-[N,N′-bis(benzyloxycarbonyl)guanido]phenylalanine-N-phenylamide.

(a)N-[4-tert-Butoxy-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-[N,N′-bis(benzyloxycarbonyl)guanido]phenylalanine-N-phenylamide

White solid (18%), m.p.; 166-169° C., specific rotation[α]_(D)=−10.5°(c=1.0, CHCl₃), Rf value; 0.21 (CHCl₃:MeOH=50:1).

¹H-NMR(CDCl₃) δ ppm; 0.6-0.9 (6H, m, (CH₃)CH₂ CH—CH₂), 0.9-1.9 (16H,s+m, (CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph+C(CH₃)₃ ), 2.2-2.6 (4H, m,CH—CO×2+Ph—CH₂ , 3.09 (2H, m, CH₂ —C₆H₄), 4.6-5.0 (2H, m, NH—CH—CO+NH),5.0-5.3 (4H, m, Ph—CH₂ —O×2), 6.6-7.7 (27H, m, aromatic-H+NH×3).

(b)N-[4-(N-benzyloxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltrimethylene)succinyl]-L-4′-[N,N′-bis(benzyloxycarbonyl)guanido]phenylalanine-N-phenylamide

White solid (38%), m.p.; 240° C. (dec.), specific rotation[α]_(D)=+3.42° (c=1.1, DMF), Rf value; 0.32 (CHCl₃:MeOH=20:1).

¹H-NMR (DMSO-d₆) δ ppm; 0.3-0.95 (6H, m, (CH₃)₂ CH—CH₂), 1.0-1.65 (7H,m, (CH₃)₂ CH—CH₂ +CH₂—CH₂ —CH₂—Ph), 1.7-2.4 (4H, m, CH—CO×2+Ph—CH₂ ),2.6-3.2 (2H, m, CH₂ —C₆H₄), 4.5-4.95 (3H, s+m, NH—CH—CO+Ph—CH₂ —O),5.0-5.4 (4H, m, Ph—CH₂ —O×2), 6.8-7.8 (29H, m, aromatic-H).

(c)N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(RS)-(3-phenyltriraethylene)succinyl]-L-4′-guanidophenylalanine-N-phenylamideMonoacetate

White amorphous powder (96%), specific rotation[α]_(D)=−11.1° (c=1.0,MeOH), Rf value; 0.32 and 0.36 (CHCl₃:MeOH:AcOH=5:2:1). Analytical valuecalculated for C₃₅H₄₆N₆O₆. Theoretical value:C, 65; H, 7.17; N, 12.99.Found:C, 65.19; H, 7.18; N, 12.95.

¹H-NMR (MeOH-d₄) δ ppm; 0.5-1.8 (13H, m, (CH₃)₂CH—CH₂ +CH₂—CH₂ —CH₂—Ph),1.92 (3H, s, CH₃ CO₂H), 2.0-2.8 (4H, m, CH—CO×2+Ph—CH₂ ), 2.8-3.3 (2H,m, CH₂ —C₆H₄), 4.5-5.2 (1H, m, NH—CH—CO), 6.8-7.8 (14H, m, aromatic-H).

EXAMPLE 20 Assay for Collagenase Inhibitory Activity

Inhibitory activities for collagenase (MMP-1) of the compounds ofExamples and comparative compounds were measured by the method of Y.Murawaki et al., [Journal of Hepatology, 18, p. 328-334 (1993)]. Thelatent procollagenase was activated by incubating with mercuryaminophenylacetate (2 mM) at 35° C. for 2 hours. The assay was done byusing bovine type I collagen labeled with fluorescein as a substrate.The activated collagenase was added to the substrate solution (0.5mg/ml) in Tris HCl buffer (50 mM, pH 7.5) containing sodium chloride(0.4 M) and calcium chloride (10 mM). The obtained solution wasincubated at 35° C. for 2 hours. The enzyme digestion was stopped by theaddition of o-phenanthroline (80 mM). The mixture was added with pigspleen elastase solution (25 μg/ml) in the above Tris HCl buffer, andincubated 37° C. for 10 min. The obtained solution was added with 70%ethanol and Tris HCl buffer (170 mM, pH 9.5) containing sodium chloride(0.61 M), whereby the indigested substrate was sedimented bycentrifuging at 3000 g for 20 min. The supernatant was taken, and itsintensity of fluorescence was determined by the excitation wavelength(495 nm) and the measured wavelength (520 nm), and the inhibitoryactivity was obtained. IC₅₀ is a concentration of a compound assayed inthe digesting enzyme, whereby the splitting of the substrate wasdecreased to 50% compared with that attained by the enzyme alone. Themeasurement results are shown in Table 1. Each compound showed equal orstronger inhibitory activity compared with the comparative compound(Table 1). Further, the comparative compounds areN-[4-(N-Hydroxyamino)-2(R)-isobutylsuccinyl]-O-methyl-L-tyrosine-N-methylamide(comparative compound 1, U.S. Pat. No. 4599361),N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-phenyltrimethylenesuccinyl]-L-tyrosine-N-methylamide(comparative compound 2, EP No. 575844 A2),N-[4-(N-Hydroxyamino)-2(R)-isobutyl-3(S)-thienyltiomethylenesuccinyl]-L-phenylalanine-N-methylamide(comparative compound 3, U.S. Pat. No. 5,412,145) andN-[4-(N-Hydroxyamino)-2(R)-n-propyloxymethylene-3(S)-isopropylthiomethylenesuccinyl]-O-methyl-L-tyrosine-N-methylamide(comparative compound 4, U.S. Pat. No. 5,442,110), which weresynthesized by referring to each patent.

TABLE 1 Inhibitory activities for matrix metalloproeinases (MMPs) (IC₅₀value, M) Compound MMP-1 MMP-3 Example 1 (Isomer B) 5.1 × 10⁻⁹ 5.0 ×10⁻⁷ Example 2 4.2 × 10⁻⁹ 2.0 × 10⁻⁷ Example 9 1.7 × 10⁻⁸ 4.0 × 10⁻⁷Comparative Compound 1 4.0 × 10⁻⁹ 1.1 × 10⁻⁷ Example 3 (Isomer B) 4.0 ×10⁻¹⁰ 5.4 × 10⁻⁹ Example 7 8.0 × 10⁻⁹ 1.0 × 10⁻⁸ Example 8 6.0 × 10⁻⁹6.0 × 10⁻⁹ Example 10 7.0 × 10⁻⁹ 1.5 × 10⁻⁸ Example 11 5.8 × 10⁻⁹ 9.0 ×10⁻⁹ Example 12 1.0 × 10⁻⁸ 1.0 × 10⁻⁸ Example 13 4.0 × 10⁻⁹ 1.0 × 10⁻⁸Example 14 4.0 × 10⁻⁹ 1.0 × 10⁻⁸ Example 15 8.0 × 10⁻⁹ 5.8 × 10⁻⁹Example 17 5.0 × 10⁻⁹ 5.0 × 10⁻⁹ Example 18 5.0 × 10⁻⁷ 6.0 × 10⁻⁹Example 19 8.0 × 10⁻⁹ 1.0 × 10⁻⁸ Comparative Compound 2 1.5 × 10⁻¹⁰ 8.0× 10⁻⁹ Example 5 (Isomer A) 8.0 × 10⁻⁹ 8.0 × 10⁻⁹ Comparative Compound 34.2 × 10⁻¹⁰ 3.0 × 10⁻⁹ Example 6 2.0 × 10⁻⁸ 2.8 × 10⁻⁷ ComparativeCompound 4 1.1 × 10⁻⁸ 1.0 × 10⁻⁷

EXAMPLE 21 Assay for Stromelysin Inhibitory Activity

Inhibitory activities for stromelysin (MMP-3) of the compounds ofExamples and comparative compounds were measured by the method of S. S.Twining et al., [Anal. Biochem., 143, p. 30 (1984)]. The latentprostromelysin was incubated with human plasmin (20 μg/ml) for theactivation at 37° C. for 2 hours, and then the incubate was added withdiisopropyl fluorophosphate solution (2.8 mg/ml) to stop the reaction.The assay was done by using casein labeled with fluorescein as thesubstrate. The activated stromelysin was added to a substrate solution(1 mg/ml) in Tris HCl buffer (50 mM, pH 7.8) containing calcium chloride(10 mM). The obtained solution was incubated at37° C. for 2 hours. Theenzyme digestion forthesubstrate was stopped by adding 5%trichloroacetic acid. The indigested substrate was sedimented bycentrifuging at 3000 g for 20 min. The supernatant was taken and addedwith Tris HCl buffer (0.5 M, pH 8.5), whereby its intensity offluorescence was determined by the excitation wavelength (495 nm) andthe measured wavelength (520 nm), and the inhibitory activity wasobtained. IC₅₀ is a concentration of a compound assayed in the digestingenzyme, whereby the splitting of the substrate was decreased to 50%compared with that attained by the enzyme alone. The measurement resultsare shown in Table 1. Each compound having the same stereo-configurationas that of the comparative compound showed equal or stronger inhibitoryactivity compared with the comparative compound (Table 1).

EXAMPLE 22 Assay for Suppression of TNF-α Production

Human monocyte derived leukemia cell-line U937 [5% bovine fetal serum(Men-eki Seibutsu Ken) was added to RPMI1640 (Nissui Seiyaku) andincubated in a 5% CO₂ incubation chamber at 37° C., whereby 1×10⁶cells/1 ml were placed into each well of a multi-well plate and thenincubated in presence of Phorbol 12-myristate 13-acetate (10⁻⁷ M, WakoJunyaku) overnight and differentiated to macrophage like cells.] wasadded with Lipopoplysaccaride (0.1 μg/ml, Sigma) derived from E.coli0127:B8 strain alone or together with a compound in Examples, andincubated in a 5% CO₂ incubation chamber at 37° C. for 6 hours. Afterthe incubation, the culture medium was taken and centrifuged at 3000 gfor 10 min., whereby the supernatant was diluted with purified waterforthe measurement using the TNF-α measurement kit (Genzyme) (Table 2).

TABLE 2 Inhibitory activities for TNF-α converting enzyme Compound IC₅₀value (M) Example 4 5.1 × 10⁻⁷ Example 5 5.0 × 10⁻⁷ Example 8 9.8 × 10⁻⁸Example 11 4.0 × 10⁻⁷ Example 16 7.0 × 10⁻⁶

EXAMPLE 23 Measurement of Solubility in Water

A compound of Examples or a comparative compound was added to distilledwater and shaken vigorously at 20±5° C. for 30 seconds at 5 min.interval. After 30 min., it was checked by watching with eye whether thecompound was dissolved or not. The measurement results are shown inTable 3. The solubility of each of the comparative compounds are lessthan 1 mg/ml. On the contrary, those of the compounds in Examples arenot less than 30-320 mg/ml of water-solubility (Table 3).

TABLE 3 Solubility in water Compound mg/ml Example 1 (Isomer A) >240Example 1 (Isomer B) >240 Example 2 >250 Example 9 >200 ComparativeCompound 1   1> Example 3 (Isomer A) >100 Example 3 (Isomer B)  150Example 4  150 Example 7  100 Example 8  30 Example 15  120 ComparativeCompound 2   1> Example 5 (Isomer A) >320 Example 5 (Isomer B) >300Comparative Compound 3   1> Example 6 >120 Comparative Compound 4   1>

EXAMPLE 24 Acute Toxicity Test

A compound of Examples was dissolved in water for injection at theconcentration of 10 mg/ml. The solution was injected in a mouse tailvein in 10 mg/Kg. The behavior of mice was watched for 8 days.

In all the compounds of Examples, there was observed no example of deathand of body weight loss.

The next embodiment shows pharmaceutical preparations containing acompound of formula (I) provided by the invention.

EXAMPLE 25

Ointment containing the following ingredients could be prepared by aconventional method.

Ingredient Amount A; White vaseline 97.8 g Liquid paraffin 2 g Example 1compound (isomer B) 0.2 g Total amount 100 g B; White vaseline 97.8 gPurified lanoline 2 g Example 1 compound (isomer B) 0.2 g Total amount100 g C; White vaseline 99.8 g Example 1 compound (isomer B) 0.2 g Totalamount 100 g

EXAMPLE 26

Eye drops containing the following ingredients could be prepared by aconventional method.

Ingredient Amount A; Example 4 compound  0.1 g Sodium chloride 0.33 gPurified sterile water appropriate Total amount  100 g B; Example 4compound  0.1 g Sodium chloride 0.26 g Anhydrous sodiumdihydrogenphosphate 0.56 g Anhydrous sodium hydrogenphosphate 0.28 g0.002% Benzalkonium chloride solution appropriate Total amount  100 g C;Example 4 compound  0.1 g Sodium chloride 0.33 g Anhydrous sodiumsulfite 0.10 g Purified sterile water appropriate Total amount  100 g D;Example 4 compound  0.1 g Sodium chloride 0.33 g Purified sterile waterappropriate Total amount  100 g

EXAMPLE 27

Injections containing the following ingredients could be prepared by aconventional method.

Ingredient Amount A; Example 4 compound  0.2 g Distilled water forinjection appropriate Total amount 100 g B; Example 4 compound  0.2 gSodium chloride  0.9 g Distilled water for injection appropriate Totalamount 100 g C; Example 4 compound  0.2 g Anhydrous sodiumdihydrogenphosphate   28 mg Anhydrous sodium hydrogenphosphate   167 mgSodium chloride  0.9 g Distilled water for injection appropriate Totalamount 100 g

What is claimed is:
 1. Compounds of the general formula (III)

wherein, R³ is a hydrogen atom, or a (C₁-C₆)alkyl, aryl oraryl(C₁-C₆)alkylene group, R⁴ is a hydrogen atom, or a (C₁-C₆)alkyl,aryl, aryl(C₁-C₆)alkylene or p-methoxyphenyl group, D is a hydrogenatom, a hydroxyl, protected phosphoric acid, protecteddiphosphonomethine, protected dicarboxymethine, nitro, amino, protectedguanido, protected guanidomethylene, cyano, aminomethylene or protectedamidino group, —Y—C″ or C″ group, wherein Y is a (C₁-C₆)alkylene group,an oxygen atom, an imino group, or a (C₁-C₆)alkyleneimino group, C″ is asulfonic, phosphonic acid, amidino, (C₁-C₆)acyl, acylimidoyl,diphosphonomethine or dicarboxymethine group, with the provisio that atleast one of R³, R⁴ and D is not a hydrogen atom and that when D is ahydroxyl group and either R³ or R⁴ is a hydrogen atom, neither R³ nor R⁴is an ethyl or an n-propyl group.
 2. Compounds of the general formula(IV)

wherein, R¹ is a hydrogen atom, or a hydroxyl, aryl(C₁-C₆)alkylene or—A—SO_(n)—B group wherein A is a (C₁-C₆)alkylene group, B is a(C₁-C₆)alkyl, (C₁-C₆)acyl, aryl or heterocycle group, and n is 0, 1 or2; R² is a hydrogen atom, or a (C₁-C₄)alkyl, (C₁-C₆)alkyloxy or(C₁-C₆)alkylthio group; R³ is a hydrogen atom, or a (C₁-C₆)alkyl, arylor aryl(C₁-C₆)alkylene group; R⁴ is a hydrogen atom, or a (C₁-C₆)alkyl,aryl, aryl(C₁-C₆)alkylene or p-methoxyphenyl group; R⁷ is (C₁-C₆)alkyl,benzyl, substituted benzyl, phenacyl or 2,2,2-trichloroethyl group; D isa hydrogen atom, a hydroxyl, protected phosphoric acid, protecteddiphosphonomethine, protected dicarboxymethine, nitro, amino, protectedguanido, protected guanidomethylene, cyano, aminomethylene or protectedamidino group, —Y—C″ or C″ group, wherein Y is a (C₁-C₆)alkylene group,an oxygen atom, an imino group, or a (C₁-C₆)alkyleneimino group, C″ is asulfonic, phosphonic acid, amidino, (C₁-C₆)acyl, acylimidoyl,diphosphonomethine or dicarboxymethine group, with the proviso that whenD is a hydroxyl group, R² is an iso-propyl group and either R³ or R⁴ isa hydrogen atom, neither R³ nor R⁴ is a methyl group.
 3. Compounds ofthe general formula (V)

wherein R² is a hydrogen atom, or a (C₁-C₄)alkyl, (C₁-C₆)alkyloxy or(C₁-C₆)alkylthio group; R³ is a hydrogen atom, or a (C₁-C₆)alkyl, arylor aryl(C₁-C₆)alkylene group, R⁴ is a hydrogen atom, or a (C₁-C₆)alkyl,aryl, aryl(C₁-C₆)alkylene or p-methoxyphenyl group, D is a hydrogenatom, a hydroxyl, protected phosphoric acid, protecteddiphosphonomethine, protected dicarboxymethine, nitro, amino, protectedguanido, protected guanidomethylene, cyano, aminomethylene or protectedamidino group, —Y—C″ or C″ group, wherein Y is a (C₁-C₆)alkylene group,an oxygen atom, an imino group, or a (C₁-C₆)alkyleneimino group, C″ is asulfonic, phosphonic acid, amidino, acylimidoyl, diphosphonomethine ordicarboxymethine group.